Methods of identifying and treating poor-prognosis cancers

ABSTRACT

The present invention relates generally to methods for identifying cancer patients with a poor prognosis, and to therapeutic modalities for improving prognosis by combating metastasis and abrogating chemoresistance in cancer cells. Embodiments of the present invention provide an objective means of prognostication regarding the long-term outcome of an incident of cancer, breast cancer in particular. Therapeutic modalities include immunotherapy and anti-sense therapy. Prognosis is determined by measuring the number of copies of the metadherin gene in the patient&#39;s cells.

FIELD OF THE INVENTION

The present invention relates generally to methods for identifyingbreast cancer patients with a poor prognosis, and to therapeuticmodalities for improving prognosis by combating metastasis andabrogating chemoresistance in cancer cells.

BACKGROUND

The progression of cancer from an abnormal outgrowth to alife-threatening metastatic tumor is accompanied by a myriad of geneticand epigenetic alterations accumulated along the way. The challenge ofdistinguishing crucial drivers of metastasis from thousands ofby-stander alterations remains a major obstacle in the battle againstcancer. The turn of the century has witnessed the advent of twoparallel, but individually incomplete, genomic approaches to unravel thegenetics of cancer metastasis.

The first, based on comparative analyses of expression profiles ofcancer cell line variants with different metastasis potentials, oftenobtained by in vivo selection in animal models, has led to theidentification of several metastasis genes. However, much work remainsto be done to validate the clinical relevance of metastasis genesidentified in animal model studies.

The second approach, gene expression profiling of human tumor specimens,has enabled the identification of several poor-prognosis signatures thatare predictive of recurrence and metastasis risk in human cancers.Although different poor-prognosis signatures for the same type of canceridentified in independent studies have proven to be operationallyinterchangeable for class prediction purposes in the clinic, the lack ofgene overlap between different poor-prognosis signatures has posed amajor challenge for understanding the biological underpinnings of cancerprogression and metastasis, thereby hindering the development oftargeted therapeutics. In other words, there is evidently no such thingas a universal “poor prognosis gene.” There is therefore a need toidentify a gene signature that predicts poor prognosis across clinicalclasses.

SUMMARY OF THE INVENTION

Embodiments of the present invention provide an objective means ofprognostication regarding the long-term outcome of an incident ofcancer. In a preferred embodiment, the invention relates to breastcancer. In other embodiments, the invention relates to immunotherapy andanti-sense therapy to combat metastasis in cancer, and to inhibit thedevelopment of cells that are resistant to chemotherapeutic agents.

In one embodiment, the invention provides a method of treating breastcancer or other cancer comprising: a) providing; i) a subject suspectedof having breast cancer or other cancer, ii) an agent that inhibits anactivity of metadherin, and b) administering said agent to said subject.

The method may be used when the breast cancer or other cancer is a poorprognosis cancer, including a metastatic cancer, a chemoresistantcancer, a cancer having in it a cell that has more than 2 copies of ametadherin gene, or a cancer having in it a cell that has a metadheringene copy number greater than that of a control cell, which control cellmay be a non-cancerous cell from the subject or from a referencesubject, or from a breast cancer cell or other cancer cell from thereference subject.

In some embodiments, the agent may be selected from the group consistingof an antibody to metadherin, a metadherin antisense molecule, and asmall molecule.

In a preferred embodiment, the inventnion provides a method for makingprognosis for a subject with breast cancer or other cancer. The methodcomprises a) providing a cancer cell from said subject, b) determining,for said cell, a metadherin gene copy number, and c) assigning a poorprognosis to said subject if said copy number is greater than 2.

In one embodiment, the invention provides a method of reducing thedevelopment of chemoresistant cancer cells in a subject treated with achemotherapeutic agent, the method comprising: a) administering to saidsubject a pharmaceutically acceptable amount of said chemotherapeuticagent, and b) administering to said subject an agent selected from thegroup consisting of an antibody to metadherin, a metadherin antisensemolecule, and a small molecule.

In another embodiment, the invention provides a method of determiningvariations in the copy-number of a gene across defined populationscomprising the following steps:

-   a) calculating an expression score based on expression differences    between comparison groups for each of a plurality of genes having a    genomic position on a chromosome,-   b) ordering said expression scores based on said genomic position of    each said gene,-   c) finding and quantifying an expression pattern for each said gene    by calculating a neighborhood score for each genomic locus using a    geometry-weighted sum of expression scores for all the genes on the    chromosome,-   d) assigning a weight to each expression score based on the    proximity of each gene to the locus in consideration,-   e) estimating the statistical significance of the neighborhood    score, and 25f) identifying a region of potential copy number    alteration.

In one embodiment, the method of determining variations in thecopy-number of a gene across defined populations includes finding astretch of 20 or more continuous aberrant neighborhood scores to detecta genomic copy number alteration.

In one embodiment, the method of determining variations in thecopy-number of a gene across defined populations includes finding aneighborhood score greater than zero to detect a genomic gain.

In one embodiment, the method of determining variations in thecopy-number of a gene across defined populations includes finding aneighborhood score less than zero to detect a genomic loss.

In some embodiments, the invention provides a method of treatmentwherein the treatment agent is a combination of a known chemotherapeuticagent and an antibody that binds to metadherin, or an antisense molecule(which may be, without limitation, an shRNA or an siRNA) or a smallmolecule. In one embodiment the treatment agent is co-administered withthe chemotherapeutic agent. In another embodiment, the treatment agentis conjugated to the chemotherapeutic agent. In one embodiment, thechemotherapeutic agent is paclitaxel.

In another embodiment, the invention provides a method of screening foranti-metastatic compounds comprising a) contacting a cancer cellexpressing metadherin with a test compound; and b) determining thelikelihood of said cancer cell to metastasize based on the level ofbiological activity of metadherin in the presence of said test compoundrelative to the level in the absence of said test compound. In oneembodiment, the metadherin-expressing cancer cell is in an organism,which may be a human or a non-human mammal.

In one embodiment, the invention provides a method of determining, basedon the copy number of the gene that encodes metadherin in a cell of acancer, a subject's probability of surviving that cancer.

In one embodiment, the copy number is determined in situ. In anotherembodiment, the copy number is determined in vitro. In alternativeembodiments, the copy number is determined by fluorescent in situhybridization (“FISH”), comparative genomic hybridization (CGH), highdensity single nucleotide polymorphism (SNP) genotyping, or real-timePCR.

In still other embodiments, the invention uses the aforementionedantibodies in a method of treating a cancer susceptible to treatmentwith a chemotherapeutic agent in a subject. The method comprises (a)administering to the subject a pharmaceutically acceptable amount of thechemotherapeutic agent, and (b) administering also to the subject any ofan agent that inhibits an activity of metadherin. In some embodiments,administering to the subject a pharmaceutically acceptable amount of thechemotherapeutic agent, and administering also the aforementioned agentprovides a method of reducing the development of chemoresistant cancercells in a subject treated with a chemotherapeutic agent.

In one embodiment, the invention provides a method of determining aprognosis in an individual with cancer, said cancers including but notlimited to, liver cancer, prostate cancer, and brain cancer. In someembodiments, metadherin is aberrantly expressed in said cancers. In someembodiments, an antibody molecule specific for metadherin is adminsteredto an individual at a sufficient dose such that metadherin is detectedin said individual. Antibody mediated prognosis of cancer in humanbeings is well-known in the art, for example in U.S. Pat. No. 5,030,559(herein incorporated by reference). In some embodiments, the inventionprovides a method of treating cancer by inhibing metadherin, saidcancers including but not limited to liver cancer, prostate cancer, andbrain cancer. In some embodiments, an antibody molecule specific formetadherin is adminstered to an individual with said cancer at asufficient dose such the metadherin protein is inhibited or the amountof metadherin protein is reduced. It is not necessary that there becomplete inhibition or reduction of metadherin protein, for the presentapplication it is sufficient for there to be some inhibition orreduction. Antibody treatment of human beings with cancer is well-knownin the art, for example in U.S. Pat. No. 5,736,137 (herein incorporatedby reference).

In some embodiments, an antisense molecule capable of recognizing andbinding metadherin RNA (including but not limited to mRNA andnon-spliced RNA) is administered to an individual with said cancer at asufficient dose such that metadherin RNA is inhibited or the amount ofmetadherin is reduced. In some embodiments, said anti-sense molecule isan siRNA, shRNA, and/or RNAi molecule. It is not necessary that there becomplete inhibition or reduction, for the present application it issufficient for there to be some inhibition or reduction. Anti-sensetreatment of human beings with cancer is well-known in the art, forexample U.S. Pat. No. 7,273,855 (herein incorporated by reference).

Definitions

To facilitate the understanding of this invention a number of terms (setoff in quotation marks in this Definitions section) are defined below.Terms defined herein (unless otherwise specified) have meanings ascommonly understood by a person of ordinary skill in the areas relevantto the present invention. As used in this specification and its appendedclaims, terms such as “a”, “an” and “the” are not intended to refer toonly a singular entity, but include the general class of which aspecific example may be used for illustration, unless the contextdictates otherwise. The terminology herein is used to describe specificembodiments of the invention, but their usage does not delimit theinvention, except as outlined in the claims.

The phrase “chosen from A, B, and C” as used herein, means selecting oneor more of A, B, C.

As used herein, absent an express indication to the contrary, the term“or” when used in the expression “A or B,” where A and B refer to acomposition, disease, product, etc., means one or the other, or both.Asused herein, the term “comprising” when placed before the recitation ofsteps in a method means that the method encompasses one or more stepsthat are additional to those expressly recited, and that the additionalone or more steps may be performed before, between, and/or after therecited steps. For example, a method comprising steps a, b, and cencompasses a method of steps a, b, x, and c, a method of steps a, b, c,and x, as well as a method of steps x, a, b, and c. Furthermore, thetenn “comprising” when placed before the recitation of steps in a methoddoes not (although it may) require sequential performance of the listedsteps, unless the context clearly dictates otherwise. For example, amethod comprising steps a, b, and c encompasses, for example, a methodof performing steps in the order of steps a, c, and b, the order ofsteps c, b, and a, and the order of steps c, a, and b, etc.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weights, reaction conditions,and so forth as used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersin the specification and claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and without limiting theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parametersdescribing the broad scope of the invention are approximations, thenumerical values in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains standarddeviations that necessarily result from the errors found in thenumerical value's testing measurements.

The term “not” when preceding, and made in reference to, anyparticularly named molecule (mRNA, etc.) or phenomenon (such asbiological activity, biochemical activity, etc.) means that only theparticularly named molecule or phenomenon is excluded.

The term “altering” and grammatical equivalents as used herein inreference to the level of any substance and/or phenomenon refers to anincrease and/or decrease in the quantity of the substance and/orphenomenon, regardless of whether the quantity is determinedobjectively, and/or subjectively.

The terms “increase,” “elevate,” “raise,” and grammatical equivalentswhen used in reference to the level of a substance and/or phenomenon ina first sample relative to a second sample, mean that the quantity ofthe substance and/or phenomenon in the first sample is higher than inthe second sample by any amount that is statistically significant usingany art-accepted statistical method of analysis. In one embodiment, theincrease may be determined subjectively, for example when a patientrefers to their subjective perception of disease symptoms, such as pain,clarity of vision, etc. In another embodiment, the quantity of thesubstance and/or phenomenon in the first sample is at least 10% greaterthan the quantity of the same substance and/or phenomenon in a secondsample. In another embodiment, the quantity of the substance and/orphenomenon in the first sample is at least 25% greater than the quantityof the same substance and/or phenomenon in a second sample. In yetanother embodiment, the quantity of the substance and/or phenomenon inthe first sample is at least 50% greater than the quantity of the samesubstance and/or phenomenon in a second sample. In a further embodiment,the quantity of the substance and/or phenomenon in the first sample isat least 75% greater than the quantity of the same substance and/orphenomenon in a second sample. In yet another embodiment, the quantityof the substance and/or phenomenon in the first sample is at least 90%greater than the quantity of the same substance and/or phenomenon in asecond sample. Alternatively, a difference may be expressed as an“n-fold” difference.

The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,” andgrammatical equivalents when used in reference to the level of asubstance and/or phenomenon in a first sample relative to a second.sample, mean that the quantity of substance and/or phenomenon in thefirst sample is lower than in the second sample by any amount that isstatistically significant using any art-accepted statistical method ofanalysis. In one embodiment, the reduction may be determinedsubjectively, for example when a patient refers to their subjectiveperception of disease symptoms, such as pain, clarity of vision, etc. Inanother embodiment, the quantity of substance and/or phenomenon in thefirst sample is at least 10% lower than the quantity of the samesubstance and/or phenomenon in a second sample. In another embodiment,the quantity of the substance and/or phenomenon in the first sample isat least 25% lower than the quantity of the same substance and/orphenomenon in a second sample. In yet another embodiment, the quantityof the substance and/or phenomenon in the first sample is at least 50%lower than the quantity of the same substance and/or phenomenon in asecond sample. In a further embodiment, the quantity of the substanceand/or phenomenon in the first sample is at least 75% lower than thequantity of the same substance and/or phenomenon in a second sample. Inyet another embodiment, the quantity of the substance and/or phenomenonin the first sample is at least 90% lower than the quantity of the samesubstance and/or phenomenon in a second sample. Alternatively, adifference may be expressed as an “n-fold” difference.

A number of terms herein relate to cancer. “Cancer” is intended hereinto encompass all forms of abnormal or improperly regulated reproductionof cells in a subject. “Subject” and “patient” are used hereininterchangeably, and a subject may be any mammal but is preferably ahuman. A “reference subject” herein refers to an individual who does nothave cancer. The “reference subject” thereby provides a basis to whichanother cell (for example a cancer cell) can be compared.).

The growth of cancer cells (“growth” herein referring generally to celldivision but also to the growth in size of masses of cells) ischaracteristically uncontrolled or inadequately controlled, as is thedeath (“apoptosis”) of such cells. Local accumulations of such cellsresult in a tumor. More broadly, and still denoting “tumors” herein areaccumulations ranging from a cluster of lymphocytes at a site ofinfection to vascularized overgrowths, both benign and malignant. A“malignant” tumor (as opposed to a “benign” tumor) herein comprisescells that tend to migrate to nearby tissues, including cells that maytravel through the circulatory system to invade or colonize tissues ororgans at considerable remove from their site of origin in the “primarytumor,” so-called herein. Metastatic cells are adapted to penetrateblood vessel wells to enter (“intravasate”) and exit (“extravasate”)blood vessels. Tumors capable of releasing such cells are also referredto herein as “metastatic.” The term is used herein also to denote anycell in such a tumor that is capable of such travel, or that is enroute, or that has established a foothold in a target tissue. Forexample, a metastatic breast cancer cell that has taken root in the lungis referred to herein as a “lung metastasis.” Metastatic cells may beidentified herein by their respective sites of origin and destination,such as “breast-to-bone metastatic.” In the target tissue, a colony ofmetastatic cells can grow into a “secondary tumor,” so called herein.

Primary tumors are thought to derive from a benign or normal cellthrough a process referred to herein as “cancer progression.” Accordingto this view, the transformation of a normal cell to a cancer cellrequires changes (usually many of them) in the cell's biochemistry. Thechanges are reflected clinically as the disease progresses throughstages. Even if a tumor is “clonogenic” (as used herein, an accumulationof the direct descendants of a parent cell), the biochemistry of theaccumulating cells changes in successive generations, both because theexpression of the genes (controlled by so-called “epigenetic” systems)of these cells becomes unstable and because the genomes themselveschange. In normal somatic cells, the genome (that is, all the genes ofan individual) is stored in the chromosomes of each cell (setting asidethe mitochondrial genome). The number of copies of any particular geneis largely invariant from cell to cell. By contrast, “genomicinstability” is characteristic of cancer progression. A genome in acancer cell can gain (“genomic gain”) or lose (“genomic loss”) genes,typically because an extra copy of an entire chromosome appears(“trisomy”) or a region of a chromosome replicates itself (“genomicgain” or, in some cases, “genomic amplification”) or drops out when thecell divides. Thus, the “copy number” of a gene or a set of genes,largely invariant among normal cells, is likely to change in cancercells (referred to herein as a “genomic event”), which affects the totalexpression of the gene or gene set and the biological behavior(“phenotype”) of descendent cells. Thus, in cancer cells, “geneactivity” herein is determined not only by the multiple “layers” ofepigenetic control systems and signals that call forth expression of thegene but by the number of times that gene appears in the genome. Theterm “epigenetic” herein refers to any process in an individual that, inoperation, affects the expression of a gene or a set of genes in thatindividual, and stands in contrast to the “genetic” processes thatgovern the inheritance of genes in successive generations of cells orindividuals.

It is thought that the emergence of metastatic cells entails its owndistinct progression, referred to herein as “metastatic progression.”The effect of disrupting a tumor on metastatic progression is unclear,but of interest because of “metastatic seeding,” herein meaning a“surge” in metastasis that occurs, for example, when a tumor issurgically resected.

Certain regions of chromosomes, depending upon the specific type ofcancer, have proven to be hot spots for genomic gain inasmuch asincreases in copy number in the genomes of cells from multiple donorstend to occur in one or a few specific regions of a specific chromosome.Such hot spots are referred to herein as sites of “recurrent genomicgain.” The term is to be distinguished from “recurrent cancer,” whichrefers to types of cancer that are likely to recur after an initialcourse of therapy, resulting in a “relapse.”

The term “prognosis,” as used herein, relates to predictions regardingthe long-term survival of cancer patients. In some contexts, the termmay be used in connection with classifying various types of cancer(e.g., likelihood of recurrence; likelihood of metastasis). In somecontexts, the term may be used in classifying particular patientssuffering from a particular clinical type of cancer. For example, twopatients having clinically identical forms of breast cancer maynevertheless not share the same prognosis with respect to the likelihoodof recurrence or the likelihood of metastasis. “Prognosis” may also bedetermined by the tendency of a cancer (either by clinical type orwithin a particular patient) to resist or develop resistance topharmaceuticals used to kill cancer cells or arrest their growth. Suchdrugs, referred herein as “chemotherapeutic” agents, include withoutlimitation doxorubicin and paclitaxel. Cancer cells susceptible to oneor another of these agents tend to adapt to the presence ofchemotherapeutic agents by becoming “chemoresistant.” Different cancers(by clinical type and within a given patient) vary in this respect.Thus, prognosis is also a function of chemoresistance. Mechanisms ofchemoresistance are incompletely understood and need not be understoodto practice embodiments of the instant invention. In general, however,the rate at which a cell takes in a drug (“drug uptake”) and the extentto which a cell retains it (“drug retention”), contribute to a cell'stendency to resist being compromised by the drug.

A number of terms herein relate to methods that enable the practitionerto examine many distinct genes at once. By these methods, sets of genes(“gene sets”) have been identified wherein each set has biologicallyrelevant and distinctive properties as a set. Devices (which may bereferred to herein as “platforms”) in which each gene in a significantpart of an entire genome is isolated and arranged in an array of spots,each spot having its own “address,” enable one to detect,quantitatively, many thousands of the genes in a cell. More precisely,these “microarrays” typically detect expressed genes (an “expressed”gene is one that is actively transmitting its unique biochemical signalto the cell in which the gene resides). Microarray data, inasmuch asthey display the expression of many genes at once, permit thepractitioner to view “gene expression profiles” in a cell and to comparethose profiles cell-to-cell to perform so-called “comparative analysesof expression profiles.” Such microarray-based “expression data” arecapable of identifying genes that are “overexpressed” (orunderexpressed) in, for example, a disease condition. An overexpressedgene may be referred to herein as having a high “expression score.”

The aforementioned methods for examining gene sets employ a number ofwell-known methods in molecular biology, to which references are madeherein. A gene is a heritable chemical code resident in, for example, acell, virus, or bacteriophage that an organism reads (decodes, decrypts,transcribes) as a template for ordering the structures of biomoleculesthat an organism synthesizes to impart regulated function to theorganism. Chemically, a gene is a heteropolymer comprised of subunits(“nucleotides”) arranged in a specific sequence. In cells, suchheteropolymers are deoxynucleic acids (“DNA”) or ribonucleic acids(“RNA”). DNA forms long strands. Characteristically, these strands occurin pairs. The first member of a pair is not identical in nucleotidesequence to the second strand, but complementary. The tendency of afirst strand to bind in this way to a complementary second strand (thetwo strands are said to “anneal” or “hybridize”), together with thetendency of individual nucleotides to line up against a single strand ina complementarily ordered manner accounts for the replication of DNA.

Experimentally, nucleotide sequences selected for their complementaritycan be made to anneal to a strand of DNA containing one or more genes. Asingle such sequence can be employed to identify the presence of aparticular gene by attaching itself to the gene. This so-called “probe”sequence is adapted to carry with it a “marker” that the investigatorcan readily detect as evidence that the probe struck a target. As usedherein, the term “marker” relates to any surrogate the artisan may useto “observe” an event or condition that is difficult or impossible todetect directly. In some contexts herein, the marker is said to “target”the condition or event. In other contexts, the condition or event isreferred to as the target for the marker. Sequences used as probes maybe quite small (e.g., “oligonucleotides” of <20 nucleotides) or quitelarge (e.g., a sequence of 100,000 nucleotides in DNA from a “bacterialartificial chromosome” or “BAC”). A BAC is a bacterial chromosome (or aportion thereof) with a “foreign” (typically, human) DNA fragmentinserted in it. BACs are employed in a technique referred to herein as“fluorescence in situ hybridization” or “FISH.” A BAC or a portion of aBAC is constructed that has (1) a sequence complementary to a region ofinterest on a chromosome and (2) a marker whose presence is discernibleby fluorescence. The chromosomes of a cell or a tissue are isolated (ona glass slide, for example) and treated with the BAC construct. Excessconstruct is washed away and the chromosomes examined microscopically tofind chromosomes or, more particularly, identifiable regions ofchromosomes that fluoresce.

Alternatively, such sequences can be delivered in pairs selected tohybridize with two specific sequences that bracket a gene sequence. Acomplementary strand of DNA then forms between the “primer pair.” In onewell-known method, the “polymerase chain reaction” or “PCR,” theformation of complementary strands can be made to occur repeatedly in anexponential amplification. A specific nucleotide sequence so amplifiedis referred to herein as the “amplicon” of that sequence. “QuantitativePCR” or “qPCR” herein refers to a version of the method that allows theartisan not only to detect the presence of a specific nucleic acidsequence but also to quantify how many copies of the sequence arepresent in a sample, at least relative to a control. As used herein,“qRTPCR” may refer to “quantitative real-time PCR,” used interchangeablywith “qPCR” as a technique for quantifying the amount of a specific DNAsequence in a sample. However, if the context so admits, the sameabbreviation may refer to “quantitative reverse transcriptase PCR,” amethod for determining the amount of messenger RNA present in a sample.Since the presence of a particular messenger RNA in a cell indicatesthat a specific gene is currently active (being expressed) in the cell,this quantitative technique finds use, for example, in gauging the levelof expression of a gene.

Collectively, the genes of an organism constitute its genome. The term“genomic DNA” may refer herein to the entirety of an organism's DNA orto the entirety of the nucleotides comprising a single gene in anorganism. A gene typically contains sequences of nucleotides devoted tocoding (“exons”), and non-coding sequences that contribute in one way oranother to the decoding process (“introns”).

The term “gene” refers to a nucleic acid (e.g., DNA) comprisingcovalently linked nucleotide monomers arranged in a particular sequencethat comprises a coding sequence necessary for the production of apolypeptide or precursor or RNA (e.g., tRNA, siRNA, rRNA, etc.). Thepolypeptide can be encoded by a full-length coding sequence or by anyportion of the coding sequence so long as the desired activities orfunctional properties (e.g., enzymatic activity, ligand binding, signaltransduction, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region together with the sequenceslocated adjacent to the coding region on both the 5′ and 3′ ends, suchthat the gene corresponds to the length of the full-length mRNA (alsoreferred to as “pre-mRNA,” “nuclear RNA,” or “primary transcript RNA”)transcribed from it. The sequences that are located 5′ of the codingregion and are present on the mRNA are referred to as 5′ untranslatedsequences. The sequences that are located 3′ or downstream of the codingregion and that are present on the mRNA are referred to as 3′untranslated sequences. The term “gene” encompasses both cDNA (thecoding region(s) only) and genomic forms of a gene. A genomic form orclone of a gene contains the coding region, which may be interruptedwith non-coding sequences termed “introns” or “intervening regions” or“intervening sequences.” Introns are removed or “spliced out” from thenuclear or primary transcript, and are therefore absent in the messengerRNA (mRNA) transcript. The mRNA functions during translation to specifythe sequence or order of amino acids in a nascent polypeptide.

Encoding in DNA (and messenger RNA) is accomplished by 3-memberednucleotide sequences called “codons.” Each codon encrypts an amino acid,and the sequence of codons encrypts the sequence of amino acids thatidentifies a particular protein. The code for a given gene is embeddedin a (usually) much longer nucleotide sequence and is distinguishable tothe cell's decoding system from the longer sequence by a “start codon”and a “stop” codon. The decoding system reads the sequence framed bythese two codons (the so-called “open reading frame”). The readable codeis transcribed into messenger RNA which itself comprises sites thatensure coherent translation of the code from nucleic acid to protein. Inparticular, the open reading frame is delimited by a so-called“translation initiation” codon and “translation termination” codon.

The term “metadherin gene” refers herein to the full-length. metadherinnucleotide sequence (e.g., contained in SEQ ID NO: XX). However, it isalso intended that the term encompass fragments of the metadherinsequence, and/or other domains within the full-length metadherinnucleotide sequence. Furthermore, the terms “metadherin nucleotidesequence” or “Metadherin polynucleotide sequence” encompasses DNA, cDNA,and RNA (e.g., mRNA) sequences.

The term “plasmid” as used herein, refers to a small, independentlyreplicating, piece of DNA. Similarly, the term “naked plasmid” refers toplasmid DNA devoid of extraneous material typically used to effecttransfection. As used herein, a “naked plasmid” refers to a plasmidsubstantially free of calcium-phosphate, DEAE-dextran, liposomes, and/orpolyamines. As used herein, the term “purified” refers to molecules(polynucleotides or polypeptides) that are removed from their naturalenvironment, isolated or separated. “Purified” molecules are at least50% free, preferably at least 75% free, and more preferably at least 90%free from other components with which they are naturally associated.

The term “recombinant DNA” refers to a DNA molecule that is comprised ofsegments of DNA joined together by means of molecular biologytechniques. Similarly, the term “recombinant protein” refers to aprotein molecule that is expressed from recombinant DNA.

The term “fusion protein” as used herein refers to a protein formed byexpression of a hybrid gene made by combining two gene sequences.Typically this is accomplished by cloning a cDNA into an expressionvector in frame (i.e., in an arrangement that the cell can transcribe asa single mRNA molecule) with an existing gene. The fusion partner mayact as a reporter (e.g., (βgal) or may provide a tool for isolationpurposes (e.g., GST).

Where an amino acid sequence is recited herein to refer to an amino acidsequence of a protein molecule, “amino acid sequence” and like terms,such as “polypeptide” or “protein” are not meant to limit the amino acidsequence to the complete, native amino acid sequence associated with therecited protein molecule. Rather the terms “amino acid sequence” and“protein” encompass partial sequences, and modified sequences.

The term “wild type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild type gene is the variant mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene

In contrast, the terms “modified,” “mutant,” and “variant” (when thecontext so admits) refer to a gene or gene product that displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Insome embodiments, the modification comprises at least one nucleotideinsertion, deletion, or substitution.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid and is referred to using the functional term “substantiallyhomologous.” The term “inhibition of binding,” when used in reference tonucleic acid binding, refers to reduction in binding caused bycompetition of homologous sequences for binding to a target sequence.The inhibition of hybridization of the completely complementary sequenceto the target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely homologous sequence to a target under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target that lacks even a partial degreeof complementarity (e.g., less than about 30% identity); in the absenceof non-specific binding the probe will not hybridize to the secondnon-complementary target.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above.

As used herein, the term “competes for binding” when used in referenceto a first and a second polypeptide means that the first polypeptidewith an activity binds to the same substrate as does the secondpolypeptide with an activity. In one embodiment, the second polypeptideis a variant of the first polypeptide (e.g., encoded by a differentallele) or a related (e.g., encoded by a homolog) or dissimilar (e.g.,encoded by a. second gene having no apparent relationship to the firstgene) polypeptide. The efficiency (e.g., kinetics or thermodynamics) ofbinding by the first polypeptide may be the same as or greater than orless than the efficiency of substrate binding by the second polypeptide.For example, the equilibrium binding constant (K_(D)) for binding to thesubstrate may be different for the two polypeptides.

As used herein, the term “hybridization” refers to the pairing ofcomplementary nucleic acids. Hybridization and the strength ofhybridization (i..e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.44(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985]). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Those skilled in the art will recognizethat “stringency” conditions may be altered by varying the parametersjust described either individually or in concert. With “high stringency”conditions, nucleic acid base pairing will occur only between nucleicacid fragments that have a high frequency of complementary basesequences (e.g., hybridization under “high stringency” conditions mayoccur between homologs with 85-100% identity, preferably 70-100%identity). With medium stringency conditions, nucleic acid base pairingwill occur between nucleic acids with an intermediate frequency ofcomplementary base sequences (e.g., hybridization under “mediumstringency” conditions may occur between homologs with 50-70% identity).Thus, conditions of “weak” or “low” stringency are often required withnucleic acids that are derived from organisms that are geneticallydiverse, as the frequency of complementary sequences is usually less.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution comprising 5×SSPE (43.8 g/l NaCl, 6.9 g/l NaH₂PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 100 to about 1000 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution comprising 5×SSPE (43.8 g/l NaCl, 6.9 g/l NaH₂PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when aprobe of about 100 to about 1000 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution comprising 5×SSPE (43.8 g/l NaCl,6.9 g/l NaH 2 PO₄ H₂ 0 and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH),0.1% SDS, 5×Denhardt's reagent [50×Denhardt's contains per 500 ml: 5 gFicoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 g/mldenatured salmon sperm DNA followed by washing in a solution comprising5×SSPE, 0.1% SDS at 42° C. when. a probe of about 100 to about 1000nucleotides in length is employed.

The term “equivalent” when made in reference to a hybridizationcondition as it relates to a hybridization condition of interest meansthat the hybridization condition and the hybridization condition ofinterest result in hybridization of nucleic acid sequences which havethe same range of percent (%) homology. For example, if a hybridizationcondition of interest results in hybridization of a first nucleic acidsequence with other nucleic acid sequences that have from 85% to 95%homology to the first nucleic acid sequence, then another hybridizationcondition is said to be equivalent to the hybridization condition ofinterest if this other hybridization condition also results inhybridization of the first nucleic acid sequence with the other nucleicacid sequences that have from 85% to 95% homology to the first nucleicacid sequence.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “sequenceidentity”, “percentage of sequence identity”, and “substantialidentity”. A “reference sequence” is a defined sequence used as a basisfor a sequence comparison; a reference sequence may be a subset of alarger sequence, for example, as a segment of a full-length cDNAsequence given in a sequence listing or may comprise a complete genesequence. Generally, a reference sequence is at least 20 nucleotides inlength, frequently at least 25 nucleotides in length, and often at least50 nucleotides in length. Since two polynucleotides may each (1)comprise a sequence (i.e., a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) mayfurther comprise a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window”, as usedherein, refers to a conceptual segment of at least 20 contiguousnucleotide positions wherein a polynucleotide sequence may be comparedto a reference sequence of at least 20 contiguous nucleotides andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman (Smithand Waterman, Adv. Appl. Math ., 2: 482, 1981) by the homology alignmentalgorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol .,48:443, 1970), by the search for similarity method of Pearson and Lipman(Pearson and Lipman, Proc. Natl. Acad. Sci., U.S.A ., 85:2444, 1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package Release 7.0,Genetics Computer Group, Madison, Wis.), or by inspection, and the bestalignment (i..e., resulting in the highest percentage of homology overthe comparison window) generated by the various methods is selected. Theterm “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 85 percentsequence identity, preferably at least 90 to 95 percent sequenceidentity, more usually at least 99 percent sequence identity as comparedto a reference sequence over a comparison window of at least 20nucleotide positions, frequently over a window of at least 25-50nucleotides, wherein the percentage of sequence identity is calculatedby comparing the reference sequence to the polynucleotide sequence whichmay include deletions or additions which total 20 percent or less of thereference sequence over the window of comparison. The reference sequencemay be a subset of a larger sequence, for example, as a segment of thefull-length sequences of the compositions claimed in the presentinvention (e.g., m.etadherin)

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having acidic side chains is glutamic acid and asparticacid; a group of amino acids having basic side chains is lysine,arginine, and histidine; and a group of amino acids havingsulfur-containing side chains is cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

“Amplification” is used herein in two different ways. A given. genetypically appears in a genome once, on one chromosome. Since chromosomesin somatic cells of eukaryotes are in general paired, two copies oralleles of each gene are found. In some conditions, such as cancer,replication of chromosome pairs during cell division is disturbed sothat multiple copies of a gene or chromosome accrue over successivegenerations. The pheonomenon is referred to generally (and herein) as“amplification.”

In the context of molecular biological experimentation, the term is useddifferently. Experimentally, “amplification” is used in relation to aspecial case of nucleic acid replication involving template specificity.It is to be contrasted with non-specific template replication (i.e.,replication that is template-dependent but not dependent on a specifictemplate). Template specificity is here distinguished from fidelity ofreplication (i.e., synthesis of the proper polynucleotide sequence) andnucleotide (ribo- or deoxyribo-) specificity. Template specificity isfrequently described in terms of “target” specificity. Target sequencesare “targets” in the sense that they are sought to be sorted out fromother nucleic acid. Amplification techniques have been designedprimarily for this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, under theconditions in which they are used, will process only specific sequencesof nucleic acids in a heterogeneous mixture of nucleic acids. Inparticular, Taq and Pfu polymerases, by virtue of their ability tofunction at high temperature, are found to display high specificity forthe sequences bounded and thus defined by the primers; the hightemperature results in thermodynamic conditions that favor primerhybridization with the target sequences and not hybridization withnon-target sequences.

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target”(defined below). In contrast, “background template” is used in referenceto nucleic acid other than sample template that may or may not bepresent in a sample. Background template is most often inadvertent. Itmay be the result of carryover, or it may be due to the presence ofnucleic acid contaminants sought to be purified away from the sample.For example, nucleic acids from .organisms other than those to bedetected may be present as background in a test sample.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particularmetadherin sequences. It is contemplated that any probe used in thepresent invention will be labelled with any “reporter molecule,” so thatis detectable in any detection system, including, but not limited toenzyme (e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label.

As used herein_(;) the term “target,” when used in reference to thepolymerase chain reaction, refers to the region of nucleic acid boundedby the primers used for polymerase chain reaction. Thus, the “target” issought to be sorted out from other nucleic acid sequences. A “segment”is defined as a region of nucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of Mullis (U.S. Pat. Nos. 4,683,195, 4,683,202, and4,965,188, hereby incorporated by reference), that describe a method forincreasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. This process foramplifying the target sequence consists of introducing a large excess oftwo oligonucleotide primers to the DNA mixture containing the desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The two primers are complementary totheir respective strands of the double stranded target sequence. Toeffect amplification, the mixture is denatured and the primers thenannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing, and polymerase extension can be repeated many times(i.e., denaturation, annealing and extension constitute one “cycle”;there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified.”

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant nucleic acid with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is present in a fonn or settingthat is different from that in which it is found in nature. In contrast,non-isolated nucleic acids are nucleic acids such as DNA and RNA foundin the state they exist in nature. For example, a given DNA sequence(e.g., a gene) is found on the host cell chromosome in proximity toneighboring genes; RNA sequences, such as a specific mRNA sequenceencoding a specific protein, are found in the cell as a mixture withnumerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding gene includes, by way of example, suchnucleic acid in cells ordinarily expressing gene where the nucleic acidis in a chromosomal location different from that of natural cells, or isotherwise flanked by a different nucleic acid sequence than that foundin nature. The isolated nucleic acid, oligonucleotide, or polynucleotidemay be present in single-stranded or double-stranded form. When anisolated nucleic acid, oligonucleotide or polynucleotide is to beutilized to express a protein, the oligonucleotide or polynucleotidewill contain at a minimum the sense or coding strand (i.e., theoligonucleotide or polynucleotide may single-stranded), but may containboth the sense and anti-sense strands (i.e., the oligonucleotide orpolynucleotide may be double-stranded).

The terms “fragment” and “portion” when used in reference to anucleotide sequence (as in “a portion of a given nucleotide sequence”)refers to partial segments of that sequence. The fragments may range insize from four nucleotides to the entire nucleotide sequence minus onenucleotide (10 nucleotides, 20, 30, 40, 50, 100, 200, etc.).

Similarly, the terms “fragment” and “portion” when used in. reference toa polypeptide sequence refers to partial segments of that sequence. Insome embodiments, the portion has an amino-terminal and/orcarboxy-terminal deletion as compared to the native protein, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the amino acid sequence deduced from a full-length cDNAsequence. Fragments are preferably at least 4 amino acids long, morepreferably at least 50 amino acids long, and most preferably at least 50amino acids long or longer (the entire amino acid sequence minus onamino acid). In particularly preferred embodiments, the portioncomprises the amino acid residues required for intermolecular binding ofthe compositions of the present invention with its various ligandsand/or substrates.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four consecutive amino acid residues tothe entire amino acid sequence minus one amino acid

As used herein the term “coding region” when used in reference tostructural gene refers to the nucleotide sequences that encode the aminoacids found in the nascent polypeptide as a result of translation of amRNA molecule. The coding region is bounded, in eukaryotes, on the 5′side by the nucleotide triplet “ATG” that encodes the initiatormethionine and on the 3′ side by one of the three triplets which specifystop codons (i.e., TAA, TAG, TGA

As used herein, the term “purified” refers to molecules (polynucleotidesor polypeptides) that are separated from other components with whichthey are naturally associated. “To purify” refers to a reduction(preferably by at least 10%, more preferably by at least 50%, and mostpreferably by at least 90%) of one or more contaminants from a sample.For example, metadherin antibodies are purified by removal ofcontaminating non-immunoglobulin proteins; they are also purified by theremoval of immunoglobulin that does not bind metadherin. The removal ofnon-immunoglobulin proteins and/or the removal of immunoglobulins thatdo not bind metadherin results in an increase in the percent ofmetadherin-reactive immunoglobulins in the sample. In another example,recombinant metadherin polypeptides are expressed in bacterial or otherhost cells and the polypeptides are purified by the removal of host cellproteins; the percent of recombinant metadherin polypeptides is therebyincreased in the sample.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques. Similarly, the term “recombinantprotein” or “recombinant polypeptide” as used herein refers to a proteinmolecule that is expressed from a recombinant DNA molecule.

The term “native protein” as used herein to indicate that a protein doesnot contain amino acid residues encoded by vector sequences, that is thenative protein contains only those amino acids found in the protein asit occurs in nature. A native protein may be produced by recombinantmeans or may be isolated from a naturally occurring source.

The term “Southern blot,” refers to the analysis of DNA on agarose oracrylamide gels to fractionate the DNA according to size followed bytransfer of the DNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized DNA is then probedwith a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists(Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, NY, pp 9.31-9.58, 1989).

The term “Northern blot,” as used herein refers to the analysis of RNAby electrophoresis of RNA on agarose gels to fractionate the RNAaccording to size followed by transfer of the RNA from the gel to asolid support, such as nitrocellulose or a nylon membrane. Theimmobilized RNA is then probed with a labeled probe to detect RNAspecies complementary to the probe used. Northern blots are a standardtool of molecular biologists (Sambrook, et al., supra, pp 7.39-7.52,1989).

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabelled antibodies

The terms “antigenic determinant” and “epitope” as used herein refer tothat portion of an antigen that makes contact with a particular antibodyand/or T cell receptor. When a protein or fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies that bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the “immunogen” used to elicitthe immune response) for binding to an antibody.

As used herein, the term “transgenic” refers to a cell or organism whosegenome has been heritably altered by genetically engineering into thegenome a gene (“transgene”) not normally part of it or removing from ita gene ordinarily present (a “knockout” gene). The “transgene” or“foreign gene” may be placed into an organism by introducing it intonewly fertilized eggs or early embryos. The term “foreign gene” refersto any nucleic acid (e.g., gene sequence) that is introduced into thegenome of an animal by experimental manipulations and may include genesequences found in that animal so long as the introduced gene does notreside in the same location as does the naturally-occurring gene.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.”

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the term host cell refers to any eukaryotic orprokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in a transgenic animal.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign. DNA failsto integrate into the genome of the transfected cell in the sense thatthe foreign DNA will be passed on to daughter cells. The termencompasses transfections of foreign DNA into the cytoplasm only. Ingeneral, however, the foreign DNA reaches the nucleus of the transfectedcell and. persists there for several days. During this time the foreignDNA is subject to the regulatory controls that govern the expression ofendogenous genes in the chromosomes. The term “transient transfectant”refers to cells that have taken up foreign DNA but have failed tointegrate this DNA. The term “transient transfection” encompassestransfection of foreign DNA into the cytoplasm only

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a cell. The uptake of nucleicacids by cells is enhanced when the nucleic acid is presented as acalcium phosphate-nucleic acid co-precipitate. The original technique ofis modified to optimize conditions for particular types of cells. Theart is well aware of these numerous modifications.

A “composition comprising a given polynucleotide sequence” as usedherein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise an aqueoussolution. Compositions comprising polynucleotide sequences encodingmetadherin or fragments thereof may be employed as hybridization probes.In this case, the metadherin-encoding polynucleotide sequences aretypically employed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS), and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

The terms “N-terminus” “NH₂ -terminus” and “amino-terminus” refer to theamino acid residue corresponding to the methionine encoded by the startcodon (e.g., position or residue 1). In contrast the terms “C-terminus”“COOH-terminus” and “carboxy terminus” refer to the amino acid residueencoded by the final codon (e.g., last or final residue prior to thestop codon).

The term “antibody” refers to polyclonal and monoclonal antibodies.Polyclonal antibodies which are formed in the animal as the result of animmunological reaction against a protein of interest or a fragmentthereof, can then be readily isolated from the blood using well-knownmethods and purified by column chromatography, for example. Monoclonalantibodies can also be prepared using known methods (See, Winter andMilstein, Nature, 349, 293-299, 1991). As used herein, the term“antibody” encompasses recombinantly prepared, and modified antibodiesand antigen-binding fragments thereof, such as chimeric antibodies,humanized antibodies, multifunctional antibodies, bispecific oroligo-specific antibodies, single-stranded antibodies and F(ab) orF(ab)₂ fragments. The term “reactive” when used in reference to anantibody indicates that the antibody is capable of binding an antigen ofinterest. For example, a metadherin antibody is an antibody which bindsto metadherin or to a fragment of metadherin.

The terms “auto-antibody” or “auto-antibodies” refer to anyimmunoglobulin that binds specifically to an antigen that is native tothe host organism that produced the antibody (i.e., the antigen is notsynthetic and/or has not been artificially supplied to the hostorganism). However, the term encompasses antibodies originally producedin response to the administration or presence of a foreign and/orsynthetic substance in the host, but also cross-react with “self”antigens. Exemplary auto-antibodies include, without limitation,anti-cholesterol ester transfer protein (CETP) antibody, anti-majorhistocompatibility complex class II antibody, anti-cytokine antibody,and anti amyloid-β-peptide antibody. The presence of auto-antibodies istermed “autoimmunity.”

The term “cytokine” refers to a molecule, such a protein orglycoprotein, involved in the regulation of cellular proliferation andfunction. Cytokines are exemplified by lymphokines (e.g., tumor necrosisfactor-α, tumor necrosis factor-β, interferon-γ, etc.), growth-factors(e.g., erythropoietin, insulin, G-CSF, M-CSF, GM-CSF, EGF, PDGF, FGF,etc.), and interleukins (e.g., IL-2, IL-4, IL-5, IL-6, IL-9, IL-10,IL-13, etc.)

The term “B cell epitope” as used herein refers to an antigenicdeterminant (protein or carbohydrate) to which a single antibodymolecule binds. B cell epitopes may comprise linear epitopes (aminoacids adjacent to each other in the primary sequence) or conformationalepitopes (moieties distant from each other in the primary sequence, butwhich are brought in proximity to one another during folding of theantigen) of at least four amino acid residues.

The term “T cell epitope” as used herein refers to an antigenicdeterminant presented by a MHC class I or class II molecule for bindingto a single T cell receptor. T cell epitopes are linear epitopescomprising at least seven amino acid residues. In some embodiments ofthe present invention, the term T cell epitope comprises a T helper cellepitope which is an antigen fragment presented by an MHC class IImolecule for binding to T cell receptor on the surface of a helper Tcell (e.g., generally CD4⁺).

The term “conservative substitution” as used herein refers to a changethat takes place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids can be divided intofour families: (1) acidic (aspartate, glutamate); (2) basic (lysine,arginine, histidine); (3) nonpolar (alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); and (4)uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3)aliphatic (glycine, alanine, valine, leucine, isoleucine, serine,threonine), with serine and threonine optionally be grouped separatelyas aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine). Whether a change in theamino acid sequence of a peptide results in a functional homolog can bereadily determined by assessing the ability of the variant peptide tofunction in a fashion similar to the wild-type protein. Peptides havingmore than one replacement can readily be tested in the same manner. Incontrast, the term “nonconservative substitution” refers to a change inwhich an amino acid from one family is replaced with an amino acid fromanother family (e.g., replacement of a glycine with a tryptophan).Guidance in determining which amino acid residues can be substituted,inserted, or deleted without abolishing biological activity can be foundusing computer programs (e.g., LASERGENE software, DNASTAR Inc.,Madison, Wis.

The terms “antigen,” “immunogen,” “antigenic,” “immunogenic,”“antigenically active,” and “immunologically active” refer to anysubstance that is capable of inducing a specific humoral and/orcell-mediated immune response. An immunogen generally contains at leastone epitope. Immunogens are exemplified by, but not restricted tomolecules which contain a peptide, polysaccharide, nucleic acidsequence, and/or lipid. Complexes of peptides with lipids,polysaccharides, or with nucleic acid sequences are also contemplated,including (without limitation) glycopeptide, lipopeptide, glycolipid,etc. These complexes are particularly useful immunogens where smallermolecules with few epitopes do not stimulate a satisfactory immuneresponse by themselves.

A peptide sequence and nucleotide sequence may be “endogenous” or“heterologous” (i..e., “foreign”). The term “endogenous” refers to asequence which is naturally found in the cell or virus into which it isintroduced so long as it does not contain some modification relative tothe naturally-occurring sequence. The term “heterologous” refers to asequence which is not endogenous to the cell or virus into which it isintroduced. For example, heterologous DNA includes a nucleotide sequencewhich is ligated to, or is manipulated to become ligated to, a nucleicacid sequence to which it is not ligated in nature, or to which it isligated at a different location in nature. Heterologous DNA alsoincludes a nucleotide sequence which is naturally found in the cell orvirus into which it is introduced and which contains some modificationrelative to the naturally-occurring sequence. Generally, although notnecessarily, heterologous DNA encodes heterologous RNA and heterologousproteins that are not normally produced by the cell or virus into whichit is introduced. Examples of heterologous DNA include reporter genes,transcriptional and translational regulatory sequences, DNA sequenceswhich encode selectable marker proteins (e.g., proteins which conferdrug resistance), etc. In preferred embodiments, the terms “heterologousantigen” and “heterologous sequence” refer to a non-hepadna virusantigen or amino acid sequence including but not limited to microbialantigens, mammalian antigens and allergen antigens.

The terms “peptide,” “peptide sequence,” “amino acid sequence,”“polypeptide,” and “polypeptide sequence” are used interchangeablyherein to refer to at least two amino acids or amino acid analogs whichare covalently linked by a peptide bond or an analog of a peptide bond.The term peptide includes oligomers and polymers of amino acids or aminoacid analogs. The term peptide also includes molecules which arecommonly referred to as peptides, which generally contain from about two(2) to about twenty (20) amino acids. The term peptide also includesmolecules which are commonly referred to as polypeptides, whichgenerally contain from about twenty (20) to about fifty amino acids(50). The term peptide also includes molecules which are commonlyreferred to as proteins, which generally contain from about fifty (50)to about three thousand (3000) amino acids. The amino acids of thepeptide may be L-amino acids or D-amino acids. A peptide, polypeptide orprotein may be synthetic, recombinant or naturally occurring. Asynthetic peptide is a peptide which is produced by artificial means invitro

The terms “oligosaccharide” and “OS” antigen refer to a carbohydratecomprising up to ten component sugars, either O or N linked to the nextsugar. Likewise, the terms “polysaccharide” and “PS” antigen refer topolymers of more than ten monosaccharide residues linked glycosidicallyin branched or unbranched chains

As used herein, the term “mammalian sequence” refers to synthetic,recombiant or purified sequences (preferably sequence fragmentscomprising at least one B cell epitope) of a mammal. Exemplary mammaliansequences include cytokine sequence, MHC class I heavy chain sequences,MHC class II alpha and beta chain sequences, and amyloid β-peptidesequences.

The terms “mammals” and “mammalian” refer animals of the class mammaliawhich nourish their young by fluid secreted from mammary glands of themother, including human beings. The class “mammalian” includes placentalanimals, marsupial animals, and monotrematal animals. An exemplary“mammal” may be a rodent, primate (including simian and human) ovine,bovine, ruminant, lagomorph, porcine, caprine, equine, canine, feline,ave, etc. Preferred non-human animals are selected from the orderRodentia.

Preferred embodiments of the present invention are primarily directed tovertebrate (backbone or notochord) members of the animal kingdom.

The terms “patient” and “subject” refer to a mammal that may be treatedusing the methods of the present invention.

The term “control” refers to subjects or samples which provide a basisfor comparison for experimental subjects or samples. For instance, theuse of control subjects or samples permits determinations to be maderegarding the efficacy of experimental procedures. In some embodiments,the term “control subject” refers to a subject that which receives amock treatment (e.g., saline alone or metadherin without a heterologousantigen insert or conjugate).

As used herein, the term “immune response” refers to the reactivity ofan organism's immune system in response to an antigen. In vertebrates,this may involve antibody production, induction of cell-mediatedimmunity, and/or complement activation (e.g., phenomena associated withthe vertebrate immune system's prevention and resolution of infection bymicroorganisms). In preferred embodiments, the term immune responseencompasses but is not limited to one or more of a “lymphocyteproliferative response,” a “cytokine response,” and an “antibodyresponse.”

The term' “antibody response” refers to the production of antibodies(e.g., IgM, IgA, IgG) which bind to an antigen of interest, thisresponse is measured for instance by assaying sera by antigen ELISA.

The term “adjuvant” as used herein refers to any compound which, wheninjected together with an antigen, non-specifically enhances the immuneresponse to that antigen. Exemplary adjuvants include but are notlimited to incomplete Freunds adjuvant (IFA), aluminum-based adjuvants(e.g., AIOH, AIPO04, etc), and Montanide ISA 720.

The terms “diluent” and “diluting agent” as used herein refer to agentsused to diminish the strength of an admixture. Exemplary diluentsinclude water, physiological saline solution, human serum albumin, oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents, antibacterial agents such as benzyl alcohol, antioxidants suchas ascorbic acid or sodium bisulphite, chelating agents such as ethylenediamine-tetra-acetic acid, buffers such as acetates, citrates orphosphates and agents for adjusting the osmolarity, such as sodiumchloride or dextrose.

The terms “carrier” and “vehicle” as used herein refer to usuallyinactive accessory substances into which a pharmaceutical substance(e.g., Metadherin vaccine) is suspended. Exemplary carriers includeliquid carriers (such as water, saline, culture medium, saline, aqueousdextrose, and glycols) and solid carriers (such as carbohydratesexemplified by starch, glucose, lactose, sucrose, and dextrans,anti-oxidants exemplified by ascorbic acid and glutathione, andhydrolyzed proteins.

The term “derived” when in reference to a peptide derived from a source(such as a microbe, cell, etc.) as used herein is intended to refer to apeptide which has been obtained (e.g., isolated, purified, etc.) fromthe source. Alternatively, or in addition, the peptide may begenetically engineered and/or chemically synthesized.

The terms “operably linked,” “in operable combination,” and “in operableorder” as used herein refer to the linkage of nucleic acid sequencessuch that they perform their intended function. For example, operablylinking a promoter sequence to a nucleotide sequence of interest refersto linking the promoter sequence and the nucleotide sequence of interestin a manner such that the promoter sequence is capable of directing thetranscription of the nucleotide sequence of interest and/or thesynthesis of a polypeptide encoded by the nucleotide sequence ofinterest. Similarly, operably linking a nucleic acid sequence encoding aprotein of interest means linking the nucleic acid sequence toregulatory and other sequences in a manner such that the protein ofinterest is expressed. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

The terms “C-terminal portion,” “COOH-terminal portion,” “carboxyterminal portion,” “C-terminal domain,” “COOH-terminal domain,” and“carboxy terminal domain,” when used in reference to an amino acidsequence of interest (such as metadherin) refer to the amino acidsequence (and portions thereof that is located from approximately themiddle of the amino acid sequence of interest to the C-terminal-mostamino acid residue of the sequence of interest. The terms “specificbinding,” “binding specificity,” and grammatical equivalents thereofwhen made in reference to the binding of a first molecule (such as apolypeptide, glycoprotein, nucleic acid sequence, etc.) to a secondmolecule (such as a polypeptide, glycoprotein, nucleic acid sequence,etc.) refer to the preferential interaction between the first moleculewith the second molecule as compared to the interaction between thesecond molecule with a third molecule. Specific binding is a relativeterm that does not require absolute specificity of binding; in otherwords, the term “specific binding” does not require that the secondmolecule interact with the first molecule in the absence of aninteraction between the second molecule and the third molecule. Rather,it is sufficient that the level of interaction between the firstmolecule and the second molecule is higher than the level of interactionbetween the second molecule with the third molecule. “Specific binding”of a first molecule with a second molecule also means that theinteraction between the first molecule and the second molecule isdependent upon the presence of a particular structure on or within thefirst molecule; in other words the second molecule is recognizing andbinding to a specific structure on or within the first molecule ratherthan to nucleic acids or to molecules in general. For example, if asecond molecule is specific for structure “A” that is on or within afirst molecule, the presence of a third nucleic acid sequence containingstructure A will reduce the amount of the second molecule which is boundto the first molecule.

For example, the term “has the biological activity of a specificallynamed protein” (such as “metadherin”) when made in reference to thebiological activity of a variant of the specifically named proteinrefers, for example, to a quantity of binding of an antibody that isspecific for the specifically named protein to the variant which ispreferably greater than 50% (preferably from 50% to 500%, morepreferably from 50% to 200%, most preferably from 50% to 100%), ascompared to the quantity of binding of the same antibody to thespecifically named protein.

Reference herein to any specifically named nucleotide sequence (such asa sequence encoding metadherin) includes within its scope fragments,homologs, and sequences that hybridize under stringent condition to thespecifically named nucleotide sequence. The term “homolog” of aspecifically named nucleotide sequence refers to an oligonucleotidesequence which exhibits greater than or equal to 50% identity to thesequence of interest. Alternatively, or in addition, a homolog of anyspecifically named nucleotide sequence (such as a sequence encodingMetadherin, a sequence encoding GSHcAg, and a sequence encoding HBcAg,etc.) is defined as an oligonucleotide sequence which has at least 95%identity with the sequence of the nucleotide sequence in issue. Inanother embodiment, the sequence of the homolog has at least 90%identity, and preferably at least 85% identity with the sequence of thenucleotide sequence in issue.

Exons, introns, genes and entire gene-sets are characteristicallylocatable with respect to one another. That is, they have generallyinvariant “genomic loci” or “genomic positions.” Genes distributedacross one or several chromosomes can be mapped to specific locations onspecific chromosomes. The field of “cytogenetics” addresses severalaspects of gene mapping. First, optical microscopy reveals features ofchromosomes that are useful as addresses for genes. In humans,chromosomes are morphologically distinguishable from one another andeach (except for the Y-chromosome) has two distinct arms separated by a“centromere.” Each arm has distinctive “bands” occupied by specificgenes. Metadherin, for example, a gene of particular interest herein, islocated on the long arm (“q”) of chromosome 8 in band 22.Disease-related changes in chromosome number, and changes in bandingform the basis for diagnosing a number of diseases. “Microdissection” ofchromosomes and DNA analysis of the microdissected fragments haveconnected specific DNA sequences to specific locations on chromosomes.In cancer, a region of a chromosome may duplicate or amplify itself ordrop out entirely. FISH, mentioned above, and “comparative genomichybridization” (“CGH”) have extended the reach of cytogenetic analysisto the extent of measuring genome alterations within and betweenindividuals. CGH, for example, in which chromosomes from a normal cellare hybridized with a corresponding preparation from a cancer cellprovides a means of directly determining cancer-related differences incopy number of chromosomal regions.

A number of terms used herein relate to antibodies. Antibodies areglobular proteins produced by cells of the immune system(“immunoglobulins”). A population of antibodies that all arose from onecell and its progeny is a “monoclonal antibody.” Others are“polyclonal.” Antibodies bind antigens. Antigens are compositions towhich an immune system has adapted by acquiring the ability tosynthesize an immunoglobulin that specifically binds to a given antigenin the sense that a “bound” antigen is no longer thermodynamically freein solution. Fragments of an antibody are capable of binding a(specific) antigen, and such fragments (e.g., Fv, Fab, Fab′ and F(ab′)₂)may be used in embodiments of the invention. Monoclonal antibodies arepreferably produced in cells maintained and reproduced in vitro. Suchcells are preferably hybridomas. Methods well known in the art are usedto create hybridoma cells, a characteristic of which is to secrete aspecific monoclonal antibody in quantity. Briefly, to create a hybridomacell line (a “cell line” herein is any collection of cells proliferatedin vitro), a mammal is immunized with the antigenic composition bound toa carrier. The carrier (e.g., protein, peptide, such as serum albumin orgamma globulin obtained from the mammal) is not recognized as a foreignmolecule to the mammal. Preferably, however, the carrier is an antibodyproduced by the mammal. The carrier antibody can bind the hapten, butnot with any specificity. Since the mammal produced the carrierantibody, the mammal will not necessarily recognize the carrier antibodyas foreign and will likely produce antibodies having binding specificityonly for the hapten. Splenocytes (typically) of the mammal are fusedwith immortalized cells to produce hybridomas and the hybridoma whichproduces a monoclonal antibody or antigen binding fragment thereofhaving the particular binding specificity for the hapten is selected.“Immortalized” cells herein are cells that reproduce indefinitely whencultured in vitro.

Monoclonal antibodies may be useful therapeutically in so-called“immunotherapy.” Monoclonal antibodies typically are products ofnon-human cells and may therefore cause untoward immune responses wheninjected into human subjects. Methods of “humanizing” such antibodiesare well-known in the art, however. In one method, the cells responsiblefor producing the antibody are genetically engineered to make andsecrete a so-called “chimeric” protein. A usually small portion of sucha protein is a fragment of the monoclonal antibody and the rest is ahuman immunoglobulin. Chimeric proteins are a particular kind of “fusionprotein.” As used herein, any protein expressed by a gene (typically, arecombinant gene) comprising the genetic code for two or more generallyindependent proteins is a fusion protein.

Monoclonal antibodies also find use herein to detect particular cells,subcellular bodies, etc. by “immunostaining.” The antibody delivers astainable (or otherwise detectable) element to its antigenicdeterminant. Thus, monoclonal antibodies are useful diagnostically for acountless number of conditions, not the least of which is their use indetermining genomic changes in cancer cells.

“Targeted therapeutics” is used herein to denote any therapeuticmodality that affects only or primarily only the cells or tissuesselected (“targeted”) for treatment. A monoclonal antibody specific foran antigen expressed only by a target (if retained by the target) ishighly useful in targeted therapeutics. In the case of unwanted cellssuch as cancer cells, if the antibody doesn't induce destruction of thetarget directly, it may do so indirectly by carrying to the target, forexample, a agent coupled to the antibody. On the other hand, agents thatsuppress processes that tend to promote uncontrolled proliferation ofcells (“antineoplastic agents”) can be delivered to target sites in thismanner.

The term “agent” is used herein in its broadest sense to refer to acomposition of matter, a process or procedure, a device or apparatusemployed to exert a particular effect. By way of non-limiting example, asurgical instrument may be employed by a practitioner as an “excising”agent to remove tissue from a subject; a chemical may be used as apharmaceutical agent to remove, damage or neutralize the function of atissue, etc. Such pharmaceutical agents are said to be “anticellular.”Cells may be removed by an agent that promotes apoptosis. A variety oftoxic agents, including other cells (e.g., cytotoxic T-cell lymphocytes)and their secretions, and a plethora of chemical species, can damagecells.

The term “by-stander”, as used herein, refers to a process or eventinitiated or affected by another, causative event or process

The term “class prediction”, as used herein, refers to a method ofmaking predictions about an individual outcome for an individual of aparticular class based on historical outcomes in similarly classifiedindividuals.

The term “Cox hazard ratios”, as used herein, refers to a particularmethod of evaluating the probability of occurrence of an eventassociated with a hazardous condition as a function of the extent ofexposure to the hazardous condition.

The term “knockdown”, as used herein, refers to a method of selectivelypreventing the expression of a gene in an individual.

The term “oncogene”, as used herein, refers to any gene that regulates aprocess affecting the suppression of abnormal proliferative events.

The term “integrative genomic analysis”, as used herein, refers to anystudy of an individual's genome by analyzing data from at least twodistinct methods of genomic analysis in combination.

The term “single nucleotide polymorphism” or “SNP”, as used herein,refers to a DNA sequence variation occurring when a single nucleotide inthe genome (or other shared sequence) differs between members of aspecies or between paired chromosomes in an individual. Singlenucleotide polymorphisms may fall within coding sequences of genes,non-coding regions of genes, or in the intergenic regions between genes.Single nucleotide polymorphisms within a coding sequence will notnecessarily change the amino acid sequence of the protein that isproduced, due to degeneracy of the genetic code. A Single nucleotidepolymorphism in which both forms lead to the same polypeptide sequenceis termed synonymous (sometimes called a silent mutation)—if a differentpolypeptide sequence is produced they are non-synonymous. Singlenucleotide polymorphisms that are not in protein-coding regions maystill have consequences for gene splicing, transcription factor binding,or the sequence of non-coding RNA.

The term “algorithm”, as used herein, refers to a step-by-stepproblem-solving procedure, especially an established, recursivecomputational procedure for solving a problem in a finite number ofsteps. The bioinformatics strategy referred to as “Analysis of CNAs byExpression data” (ACE) is one example of an algorithm that detectsrecurrent DNA copy number alterations (CNAs) that affect regional geneexpression.

The term “tissue array” or “tissue microarray”, as used herein, refersto high throughput platforms for the rapid analysis of protein, RNA, orDNA molecules. These arrays can be used to validate the clinicalrelevance of potential biological targets in the development ofdiagnostics, therapeutics and to study new disease markers and genes.Tissue arrays are suitable for genomics-based diagnostic and drug targetdiscovery.

As used herein, the term “shRNA” or “short hairpin RNA” refers to asequence of ribonucleotides comprising a single-stranded RNA polymerthat makes a tight hairpin turn on itself to provide a “double-stranded”or duplexed region. shRNA can be used to silence gene expression via RNAinterference. shRNA hairpin is cleaved into short interfering RNAs(siRNA) by the cellular machinery and then bound to the RNA-inducedsilencing complex (RISC). It is believed that the complex inhibits RNAas a consequence of the complexed siRNA hybridizing to and cleaving RNAsthat match the siRNA that is bound thereto.

As used herein, the term “RNA interference” or “RNAi” refers to thesilencing or decreasing of gene expression by siRNAs. It is the processof sequence-specific, post-transcriptional gene silencing in animals andplants, initiated by siRNA that is homologous in its duplex region tothe sequence of the silenced gene. The gene may be endogenous orexogenous to the organism, present integrated into a chromosome orpresent in a transfection vector that is not integrated into the genome.The expression of the gene is either completely or partially inhibited.RNAi inhibits the gene by compromising the function of a target RNA,completely or partially. Both plants and animals mediate RNAi by theRNA-induced silencing complex (RISC); a sequence-specific,multicomponent nuclease that destroys messenger RNAs homologous to thesilencing trigger. RISC is known to contain short RNAs (approximately 22nucleotides) derived from the double-stranded RNA trigger, although theprotein components of this activity are unknown. However, the22-nucleotide RNA sequences are homologous to the target gene that isbeing suppressed. Thus, the 22-nucleotide sequences appear to serve asguide sequences to instruct a multicomponent nuclease, RISC, to destroythe specific mRNAs. Carthew has reported (Curr. Opin. Cell Biol. 13(2):244-248 (2001)) that eukaryotes silence gene expression in the presenceof dsRNA homologous to the silenced gene. Biochemical reactions thatrecapitulate this phenomenon generate RNA fragments of 21 to 23nucleotides from the double-stranded RNA. These stably associate with anRNA endonuclease, and probably serve as a discriminator to select mRNAs.Once selected, mRNAs are cleaved at sites 21 to 23 nucleotides apart.

As used herein, the term “siRNAs” refers to short interfering RNAs. Insome embodiments, siRNAs comprise a duplex, or double-stranded region,of about 18-25 nucleotides long; often siRNAs contain from about two tofour unpaired nucleotides at the 3′ end of each strand. At least onestrand of the duplex or double-stranded region of a siRNA issubstantially homologous to or substantially complementary to a targetRNA molecule. The strand complementary to a target RNA molecule is the“antisense strand”; the strand homologous to the target RNA molecule isthe “sense strand”, and is also complementary to the siRNA antisensestrand. siRNAs may also contain additional sequences; non-limitingexamples of such sequences include linking sequences, or loops, as wellas stem and other folded structures. siRNAs appear to function as keyintermediaries in triggering RNA interference in invertebrates and invertebrates, and in triggering sequence-specific RNA degradation duringposttranscriptional gene silencing in plants.

The term “xenograft”, as used herein, refers to the transfer ortransplant of a cell(s) or tissue from one species to an unlike species(or genus or family).

The term “orthotopic” or “orthotopic xenograft”, as used herein, refersto a cell or tissue transplant grafted into its normal place in thebody.

The term “fluorescent activated cell sorting” or “FACS”, as used herein,refers to a technique for counting, examining, and sorting microscopicparticles suspended in a stream of fluid. It allows simultaneousmultiparametric analysis of the physical and/or chemical characteristicsof single cells flowing through an optical and/or electronic detectionapparatus. Generally, a beam of light (usually laser light) of a singlewavelength is directed onto a hydro-dynamically focused stream of fluid.A number of detectors are aimed at the point where the stream passesthrough the light beam; one in line with the light beam (ForwardScatter, correlates to cell volume) and several perpendicular to thebeam, (Side Scatter, correlates to the inner complexity of the particleand/or surface roughness) and one or more fluorescent detectors. Eachsuspended particle passing through the beam scatters the light in someway, and fluorescent chemicals found in the particle or attached to theparticle may be excited into emitting light at a lower frequency thanthe light source. By analyzing the combinations of scattered andfluorescent light picked up by the detectors it is then possible toderive information about the physical and chemical structure of eachindividual particle.

The term “data mining”, as used herein, refers to the automated orconvenient extraction of patterns representing knowledge implicitlystored or captured in large databases, data warehouses, internetwebsites, other massive information repositories, or data streams.

The terms “overexpress”, “overexpressing” and grammatical equivalents,as used herein, refer to the production of a gene product at levels thatexceed production in normal or control cells. The term “overexpression”or “highly expressed” may be specifically used in reference to levels ofmRNA to indicate a higher level of expression than that typicallyobserved in a given tissue in a control or non-transgenic animal. Levelsof mRNA are measured using any of a number of techniques known to thoseskilled in the art including, but not limited to Northern blot analysis.Appropriate controls are included on the Northern blot to control fordifferences in the amount of RNA loaded from each tissue analyzed, theamount of 28S rRNA (an abundant RNA transcript present at essentiallythe same amount in all tissues) present in each sample can be used as ameans of normalizing or standardizing the mRNA-specific signal observedon Northern blots. Overexpression may likewise result in elevated levelsof proteins encoded by said mRNAs.

The term “laser capture microdissection” or “LCM”, as used herein,refers to a method for isolating specific cells of interest from tissuesections wherein a transparent transfer film is applied to the surfaceof a tissue section. A pulsed laser beam activates a precise spot on thetransfer film, fusing the film with the underlying cells of choice. Thetransfer film with the bonded cells is then lifted off the thin tissuesection, leaving all unwanted cells behind. This method is useful forcollecting selected cells for DNA, RNA and/or protein analyses. LCM canbe performed on a variety of tissue samples including blood smears,cytologic preparations, cultured cells and solid tissues.

The term “heatmap”, as used herein, refers to a graphical representationof data where the values obtained from a variable two-dimensional mapare represented as colors. As related to the field of molecular biology,heat maps typically represent the level of expression of multiple genesacross a number of comparable samples as obtained from a microarray.

The term “phage display”, as used herein, refers to theintegration/ligation of numerous genetic sequences from a DNA library,consisting of all coding sequences of a cell, tissue or organism libraryinto the genome of a bacteriophage (i.e. phage) for high-throughputscreening protein-protein and/or protein-DNA interactions. Using amultiple cloning site, these fragments are inserted in all threepossible reading frames to ensure that the cDNA is translated. DNAfragments are then expressed on the surface of the phage particle aspart of it coat protein. The phage gene and insert DNA hybrid is thenamplified by transforming bacterial cells (such as TG1 E. coli cells),to produce progeny phages that display the relevant protein fragment aspart of their outer coat. By immobilizing relevant DNA or proteintarget(s) to the surface of a well, a phage that displays a protein thatbinds to one of those targets on its surface will remain while othersare removed by washing. Those that remain can be eluted, used to producemore phage (by bacterial infection with helper phage) and so produce anenriched phage mixture. Phage eluted in the final step can be used toinfect a suitable bacterial host, from which the phagemids can becollected and the relevant DNA sequence excised and sequenced toidentify the relevant, interacting proteins or protein fragments.

The term “apoptosis”, as used herein, refers to a form of programmedcell death in multicellular organisms that involves a series ofbiochemical events that lead to a variety of morphological changes,including blebbing, changes to the cell membrane such as loss ofmembrane asymmetry and attachment, cell shrinkage, nuclearfragmentation, chromatin condensation, and chromosomal DNAfragmentation. Defective apoptotic processes have been implicated in anextensive variety of diseases; for example, defects in the apoptoticpathway have been implicated in diseases associated with uncontrolledcell proliferations, such as cancer.

The term “bioluminescence imaging” or “BLI”, as used herein, refers tothe noninvasive study of ongoing biological processes in livingorganisms (for example laboratory animals) using bioluminescence, theprocess of light emission in living organisms. Bioluminescence imagingutilizes native light emission from one of several organisms whichbioluminescence. The three main sources are the North American firefly,the sea pansy (and related marine organisms), and bacteria likePhotorhabdus luminescens and Vibrio fischeri. The DNA encoding theluminescent protein is incorporated into the laboratory animal eithervia a virus or by creating a transgenic animal. While the total amountof light emitted via bioluminescence is typically small and not detectedby the human eye, an ultra-sensitive CCD camera can imagebioluminescence from an external vantage point. Common applications ofBLI include in vivo studies of infection (with bioluminescentpathogens), cancer progression (using a bioluminescent cancer cellline), and reconstitution kinetics (using bioluminescent stem cells).

The term “consensus region” or “consensus sequence”, as used herein,refers to the conserved sequence motifs that show which nucleotideresidues are conserved and which nucleotide residues are variable whencomparing multiple DNA, RNA, or amino acid sequence alignments. Whencomparing the results of a multiple sequence alignment, where relatedsequences are compared to each other, and similar functional sequencemotifs are found. The consensus sequence shows which residues areconserved (are always the same), and which residues are variable. Aconsensus sequence may be a short sequence of nucleotides, which isfound several times in the genome and is thought to play the same rolein its different locations. For example, many transcription factorsrecognize particular consensus sequences in the promoters of the genesthey regulate. In the same way restriction enzymes usually havepalindromic consensus sequences, usually corresponding to the site wherethey cut the DNA. Splice sites (sequences immediately surrounding theexon-intron boundaries) can also be considered as consensus sequences.In one aspect, a consensus sequence defines a putative DNA recognitionsite, obtained for example, by aligning all known examples of a certainrecognition site and defined as the idealized sequence that representsthe predominant base at each position. Related sites should not differfrom the consensus sequence by more than a few substitutions.

The term “seminaphtharhodafluor”, “SNARF” or “SNARF-1”, as used herein,refers to a fluorescent dye that changes color with pH, and can be usedto construct optical biosensors.

The term “linkage”, or “genetic linkage,” as used herein, refers to thephenomenon that particular genetic loci of genes are inherited jointly.The “linkage strength” refers to the probability of two genetic locibeing inherited jointly. As the distance between genetic loci increases,the loci are more likely to be separated during inheritance, and thuslinkage strength is weaker.

The term “neighborhood score”, as used herein, refers to the relativevalue assigned to a genomic locus based on a geometry-weighted sum ofexpression scores of all the genes on a given chromosome, as ameasurement of the copy number status of the locus. A positiveneighborhood score is indicative of an increase in copy number, whereasa negative neighborhood score is indicative of a decrease in copynumber.

The term “expression score”, as used herein, refers to the expressiondifferences (i.e., the level of transcription (RNA) or translation(protein)) between comparison groups on a given chromosome. Theexpression score for a given gene is calculated by correlating the levelof expression of said gene with a phenotype in comparison. For example,an expression score may represent a comparison of the expressiondifferences of a given gene in normal vs. abnormal conditions, such asparental vs. drug-resistant cell lines. As used herein, the term“regional expression score” refers to the expression score of gene(s) inproximity to the locus in consideration. Since linkage strength betweengenetic loci decreases (i.e. decays) as the distance between themincreases, the “regional expression score” more accurately reflects theexpression differences between comparison groups by assigning greaterweight to the expression scores of genes in proximity to the locus inconsideration.

The terms “geometry-weighted” or “geometry-weighted sum”, as usedherein, refers to the significance attached to a given value, forexample an “expression score”, based on physical position, including butnot limited to genomic position. Since linkage strength between geneticloci decreases (i.e. decays) as the distance between them increases, the“weight” assigned to a given value is adjusted accordingly.

The term “copy number alteration” or “CNA”, as used herein, refers tothe increase (i.e. genomic gain) or decrease (i.e. genomic loss) in thenumber of copies of a gene at a specific locus of a chromosome ascompared to the “normal” or “standard” number of copies of said genethat locus. As used herein, an increase in the number of copies of agiven gene at a specific locus may also be referred to as an“amplification” or “genomic amplification” and should not be confusedwith the use of the term “amplification” as it relates, for example, toamplification of DNA or RNA in PCR and other experimental techniques.

The term “clonogenic assay”, as used herein, refers to a technique forstudying whether a given cancer therapy (for example drugs or radiation)can reduce the clonogenic survival and proliferation of tumor cells.While any type of cell may be used, human tumor cells are commonly usedfor oncological research. The term “clonogenic” refers to the fact thatthese cells are clones of one another.

The term “adjuvant therapy”, as used herein, refers to additionaltreatment given after the primary treatment to increase the chances of acure. In some instances, adjuvant therapy is administered after surgerywhere all detectable disease has been removed, but where there remains astatistical risk of relapse due to occult disease. If known disease isleft behind following surgery, then further treatment is not technically“adjuvant”. Adjuvant therapy may include chemotherapy, radiationtherapy, hormone therapy, or biological therapy. For example,radiotherapy or chemotherapy is commonly given as adjuvant treatmentafter surgery for a breast cancer. Oncologists use statistical evidenceto assess the risk of disease relapse before deciding on the specificadjuvant therapy. The aim of adjuvant treatment is to improvedisease-specific and overall survival. Because the treatment isessentially for a risk, rather than for provable disease, it is acceptedthat a proportion of patients who receive adjuvant therapy will alreadyhave been cured by their primary surgery. Adjuvant chemotherapy andradiotherapy are often given following surgery for many types of cancer,including colon cancer, lung cancer, pancreatic cancer, breast cancer,prostate cancer, and some gynecological cancers.

The term “matched samples”, as used herein, as for example “matchedcancer samples” refers to a sample in which individual members of thesample are matched with every other sample by reference to a particularvariable or quality other than the variable or quality immediately underinvestigation. Comparison of dissimilar groups based on specifiedcharacteristics is intended to reduce bias and the possible effects ofother variables. Matching may be on an individual (matched pairs) or agroup-wide basis.

The term “genomic segments”, as used herein, refers to any defined partor region of a chromosome, and may contain zero, one or more genes.

The term “poor prognosis”, as used herein, refers to a prospect ofrecovery from a disease, infection, or medical condition that isassociated with a diminished likelihood of a positive outcome. Inrelation to a disease such as cancer, a “poor prognosis” may beassociated with a reduced patient survival rate, reduced patientsurvival time, higher likelihood of metastatic progression of saidcancer cells, and/or higher likelihood of chemoresistance of said cancercells.

The term “chemoresistant”, as used herein, refers to a cancer and/ortumor that is measurably less responsive to chemotherapeutic agents thanother cancers and/or tumors.

The term “co-administer”, as used herein, refers to the administrationof two or more agents, drugs, and/or compounds together (i.e. at thesame time).

The term “diagnose” or “diagnosis”, as used herein, refers to thedetermination, recognition, or identification of the nature, cause, ormanifestation of a condition based on signs, symptoms, and/or laboratoryfindings.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and tables.

FIG. 1 depicts the use of ACE analysis to identify recurrent genomicgain at 8q22 in poor-prognosis breast cancer.

FIG. 2 depicts validation of the ACE algorithm using availableexpression data with corresponding genomic alteration data.

FIG. 3 depicts the validation of 8q22 amplification in human breasttumors.

FIG. 4 depicts DNA copy number quantification by FISH and genomic DNAqPCR.

FIG. 5 demonstrates that MTDH mediates lung metastasis of human breastcancer.

FIG. 6 demonstrates that overexpression analysis of 8q22 genesidentified MTDH as the target gene of the amplicon to promotemetastasis.

FIG. 7 demonstrates organ-specific metastasis mediated by MTDH.

FIG. 8 demonstrates that MTDH does not influence the growth, migrationor invasion of tumor cells.

FIG. 9 demonstrates that MTDH enhances chemoresistance of breast cancercells.

FIG. 10 depicts the correlation of 8q22 copy number in NS and NC160 celllines.

FIG. 11 depicts an in vivo chemoresistance assay with doxorubicin.

FIG. 12 demonstrates that ALDH3A1 and MET contribute to MTDH-mediatedchemoresistance.

FIG. 13 depicts drug uptake and retention in cells with modified MTDHexpression.

FIG. 14 demonstrates that MTDH is associated with poor-prognosis ofhuman breast tumors.

FIG. 15 demonstrates that CCNE2 is not associated with clinical outcomesin the breast cancer and tissue array analyses.

Table 1 depicts a recurrent region of gain associated withpoor-prognosis breast cancer as detected by ACE. Only the regionsdetected in at least two of the three analyzed datasets are shown.

Table 2 depicts all poor-prognosis-associated CAN regions detected byACE in breast cancer.

Table 3 depicts Cox hazard ratios for relapse based on neighborhoodscores of each of the common regions of gain in the three publisheddatasets.

Table 4 depicts regions detected by ACE in bladder tumors compared tonormal samples.

Table 5 depicts genomic DNA copy number by qPCR, as well as theexpression by qRT-PCR or immunostaining of the genes at 8q22 in humanbreast tumor samples.

Table 6 depicts patient records of tumors used in the breast cancertissue array.

Table 7 depicts microarray data of the genes with altered expressionafter MTDH knockdown in LM2 cancer cells.

Table 8 shows Cox hazard ratios for metastasis in breast cancer based onMTDH expression levels in tissue array analysis.

Table 9 depicts the primers used in the qPCR for DNA copy number andgene expression analysis.

Table 10 lists chemotherapeutic agents.

DETAILED DESCRIPTION OF THE INVENTION

Recurrent DNA copy number alterations (CNAs) have been observed in awide range of human cancers. Such genetic events often indicate thepresence of key mediators of malignancy in the affected genomic loci.For example, elevated expression of oncogenes, such as c-Myc, CCND1,Her2 and EGFR1²²⁻²⁶, often result from amplification of correspondinggenomic segments. However, CNAs responsible for cancer metastasis arepoorly characterized. Various techniques have been developed to detectgenomic alterations, including fluorescence in situ hybridization(FISH), comparative genomic hybridization (CGH) and high-density singlenucleotide polymorphism (SNP) genotyping²⁷⁻³. Detection of CNAs byexpression profiling analysis is theoretically possible since a strongcorrelation between genomic alterations and aberrant expression of genesin affected loci has been observed³¹. Accurate detection of CNAs usingexpression analysis, however, is technically difficult because geneexpression data reflect multiple layers of gene regulation beyondgenomic alterations. Such analysis is particularly challenging withclinical tumor samples due to the inherent heterogeneity of clinicalspecimens and the rampant genomic instability of late stage tumors.

Copy number is readily determined by any of several methods well-knownin the art. Garcia et al. (U.S. Patent Application Publication2008/0090233, incorporated herein in its entirety, with citedreferences, for all purposes) have utilized FISH in particular toevaluate copy number of the epidermal growth factor receptor in cells.The method described therein is readily adapted for metadherin bypersons having ordinary skill in the art. Comparative genomichybridization (“CGH”), described, for example, in U.S. Pat. No.6,159,685 and related applications (also incorporated herein, with citedreferences, for all purposes), is another well-known method fordetermining the copy number of a gene. Together with the informationdisclosed herein, CGH is also readily adapted for use in determining thecopy number of metadherin. A variety of methods based on PCR may also beadapted for the purpose. A non-limiting example is found in U.S. Pat.No. 6,180,349, incorporated herein in its entirety by reference, whereinreal-time fluorescence PCR is employed to measure copy number. Morerecently, a method for quantifying gene copy number of individual genes,whole chromosomes or portions of chromosomes in a homogeneous reactionthat does not require amplification of the target, resolution offragment sizes, or microscopy has been described (U.S. PatentApplication Publication 2007/0087345, incorporated herein in itsentirety, with cited references, for all purposes).

In one embodiment, the present invention contemplates a computationalalgorithm termed “Analysis of CNAs by Expression data”(ACE) to identifya recurrent 8q22 genomic gain in poor-prognosis human cancers, and inparticular poor prognosis breast cancer. The 8q22 locus harbors themetastasis gene Metadherin (MTDH; also called Lyric, AEG1³²⁻³⁴). Genomicgain of 8q22 and the concurrent overexpression of MTDH were observed ina significant proportion of human primary breast tumors and wereassociated with poor survival and a higher risk of metastaticprogression. Functional characterization of MTDH in animal models and invitro functional assays revealed its dual functions in promotingmetastasis and chemoresistance of breast cancers. Inhibition (completeor partial) of MTDH expression in breast cancer cells reduced the cells'potential for metastasizing to lung and other organs, and sensitized thecells to stress and chemotherapeutic agents. Expression profiling of ahighly metastatic human breast cancer cell line LM2 revealed anMTDH-regulated gene set that includes (but is not necessarily limitedto) several genes involved in the regulation of chemosensitivity ofcancer cells to a broad spectrum of antineoplastic agents. Among thesegenes, ALDH3A1 and Met were further confirmed to play a functional rolein MTDH-mediated chemoresistance. Such results, properly integrated,uncover metastasis genes with important prognostic as well astherapeutic values, and establish MTDH as a major target for theprevention and treatment of chemoresistant metastasis.

In one embodiment, the present invention contemplates integrating suchresults by means of a computational approach to unveil functionallysignificant cytogenetic events directly linked to altered geneexpression in poor-prognosis tumors. Reasoning that metastasis genes arelikely to reside in these recurrent genomic alterations, the ACEalgorithm is designed to translate gene expression profiling data intoputative genomic alteration maps. The ACE approach has been validated inmultiple datasets, regardless of the nature of samples or the platformsof gene expression microarrays. Even in the most complicated studies ofcancer metastasis, where numerous genomic events make it difficult todetect phenotype-specific CNAs, ACE still produced reliable results thatwere validated with direct cytogenetic methods. In fact, ACE was able totake advantage of the heterogeneity of large, independent datasets topinpoint the smallest and most conserved regions of overlap that weremost likely to harbor critically important candidate metastasis genes.Although the genomic gain of different lengths near 8q22 is known tooccur in breast cancer, the phenomenon had not been clearly associatedwith metastasis and poor prognosis. More importantly, the target gene ofthis amplification event had not been identified due to the large numberof genes in this region. By analyzing multiple datasets, ACEsuccessfully narrowed the cytogenetic event to a 13-gene region thatallowed for focused functional testing of candidate genes in animalmetastasis assays.

High-throughput genomic profiling methods such as CGH and SNP arrayshave facilitated the recent discovery of several novel cancergenes^(43,53,54). As a new addition to the repertoire of integrativegenomic analysis tools, ACE is particularly useful when cytogenetic dataare not available. ACE can also be used as a complementary strategy tofine-map results obtained from cytogenetic analyses. A further advantageof ACE is that it can detect regional epigenetic alterations that cannotbe discerned by the CGH or the SNP array approach (FIG. 2d ).Additionally, ACE provides a direct link between cytogenetic events andgene activity changes, thereby facilitating the search for functionallyimportant candidate genes. In contrast, genomic alterations detected byCGH or SNP array approaches may not necessarily result in altered geneexpression. Given the large amount of archived gene expression dataavailable in public domains and the difficulty in obtaining matchedcancer samples, ACE is a useful data-mining tool to bring new insightsinto the functional mechanism of cancer progression.

ACE analysis of cancer, and in particular breast cancer, according toone embodiment of the invention, together with clinical and functionalstudies of MTDH, indicate that MTDH is a metastasis gene with prognosticpotential and therapeutic value. Brown et al.³² previously used phagedisplay to identify MTDH as a homing receptor that mediates the adhesionof the 4T1 murine mammary tumor cell line to lung endothelial cells andalso promotes lung metastasis. In that study, only the mouse 4T1 cellline and the biologically irrelevant HEK 293T cell line were used toanalyze the lung-targeting function of MTDH. The involvement of MTDH inhuman cancer, however, has not been previously reported. In addition, norigorous clinical correlation study has been performed to directly linkMTDH to human cancer metastasis, and in particular human breast cancermetastasis.

An extensive collection of human breast tumor samples analyzed accordingto an embodiment of the invention demonstrated that an elevated MTDHprotein level is an important prognostic factor independent of otherclinicopathological factors. Results indicated that a substantialproportion of human breast tumors exhibit MTDH genomic amplificationwith a subsequent increase in MTDH expression, which is associated withpoor survival and higher risk of progression.

The importance of MTDH in cancer metastasis is not necessarily limitedto promoting lung-specific spread of breast tumor cells. Indeed, thefunctional importance of MTDH in systemic metastasis using awell-established model for human breast cancer metastasis was validatedby one embodiment of the instant invention. Although MTDH was previouslyreported to enhance murine mammary tumor cell adhesion to lungendothelial cells, in this embodiment of the invention MTDH was alsoshown to enhance the affinity of human breast cancer cells for otherendothelial cell types, consistent with its role in promoting systemicmetastasis in animal models. Moreover, MTDH was aberrantly expressed intumors from liver, prostate, and brain⁵⁵⁻⁵⁷, suggesting a potentialinvolvement in a broad spectrum of cancers.

Current standard treatments for cancer, and in particular breast cancer,use the combination of surgery to remove localized disease andchemotherapy to eliminate systemic spreading. However, relapsed cancers,including breast cancer, often acquire resistance to chemotherapy andare often inoperable. Thus, over 90% of breast cancer related deaths arenot due to cancer at the primary site, but rather due to the spread ofchemoresistant cancer cells from breast to secondary vital organs, suchas lung, bone, liver and brain. Metastasis and chemoresistance remaintwo major obstacles to curative therapy. One embodiment of the presentinvention has identified MTDH as a factor in the chemoresistance ofcancer cells. Thus, MTDH may be among an important class of genes thatplay a role both in metastasis and in chemoresistance (FIG. 14e ). Thisduality may explain why some metastasis genes are selected for in theprimary tumor: whether or not they confer a growth advantage (which theytypically do not in animal tumorigenic assays), they presumably confer asurvival advantage by endowing cancer cells with enhanced tolerance totherapeutic and physiological stresses that human tumors may endure. Atthe same time, other genes at 8q22, such as SDC2 and CCNE2, may conferthe growth advantage and allow for the expansion of tumor cells with8q22 genomic gain in the primary tumor. Physical linkage ofgrowth-promoting and metastasis-driving genes in 8q22 may thus producecascading events for the expansion of the primary tumor followed by theformation of distant metastasis.

In some embodiments, microarray profiling of MTDH-knockdown cellsreveals several genes, including MET, HMOX1, ALDH3A1, and two HSP90family genes that may contribute to the chemoresistance function ofMTDH. In some embodiments, the involvement of ALDH3A1 and MET inMTDH-mediated chemoresistance is further validated by a series of invitro chemoresistance experiments in which the expression of ALDH3A1 andMET was altered in cancer cells (FIG. 12). As MTDH enhanceschemoresistance of breast cancer cells to a broad spectrum ofchemotherapeutic agents and physiological stresses, such a phenomenonmay result from the concerted actions of multiple chemoresistancemediators identified in the microarray experiment. This is consistentwith the observation that the reduction of chemoresistance is moresignificant in MTDH knockdown cells than in cells with individualknockdown of ALDH3A1 and MET, and that the effect of ALDH3A1 and METdouble knockdown reaches a level similar to that of MTDH knockdown.Although MTDH may promote metastasis by enhancing cancer cell adhesionto endothelial cells, several genes identified by the microarrayexperiment may also contribute to the pro-metastasis function of MTDH.For example, genes that are down-regulated by MTDH inhibition include(but are not necessarily limited to) several previously reportedmetastasis-promoting genes such as MET, ADAMTS1 and CTGF^(9,59,60).Conversely, several genes that have been reported to suppressmetastasis, including GPR56, TIMP3 and TRAIL⁶¹⁻⁶⁴, were overexpressed inthe MTDH-knockdown line (FIG. 12a and Table 7).

In some embodiments, a combination of computational biology, in vivo andin vitro functional metastasis assays, and extensive clinicalcorrelation analysis is used to identify an 8q22 poor-prognosis genomicgain that harbors the dual functional metastasis gene MTDH. In someembodiments, overexpression of MTDH occurs in up to 40% of breast cancerpatients and promotes metastatic seeding as well as chemoresistance ofbreast tumors. In some embodiments, this study indicates severalpotential applications in the clinical management of human cancer, andin particular poor prognosis breast cancer. In some embodiments, genomicamplification and overexpression of MTDH represent a powerful prognosismarker independent from other well-established markers for cancer, andin particular poor prognosis breast cancer. In some embodiments,molecular targeting of the dual-function metastasis gene MTDH may notonly prevent the seeding of cancer cells, and in particular poorprognosis breast cancer cells, to lung and other vital organs but alsosensitize cancer cells to chemotherapy, thereby stopping the deadlyspread of such cancers.

In some embodiments, the present invention relates to compositions andmethods for cancer diagnosis, treatment and research, including but notlimited to, cancer markers and uses of cancer markers. In particular,the present invention provides compositions and methods for targetingmetadherin in cancer, and in particular poor prognosis breast cancer.

-   I. Cancer Therapies

In some embodiments, the present invention provides therapies for cancer(e.g., breast cancer, and in particular poor prognosis breast cancer).In some embodiments, therapies target metadherin. That is, in suchembodiments, therapeutic methods are directed at reducing metadherin'sactivity by one means or another, and may be referred to herein as“anti-metadherin therapy.” It is not intended that an antimetadherintherapy be identified by any particular effect, such as reducing tumorburden, metastasis or angiogenesis. The objective of anti-metadherintherapy as the term is used herein is to promote the survival of thecancer patient. As described herein, studies conducted during the courseof development of the present invention demonstrated a role formetadherin in cancer metastasis. Further studies demonstrated thatinterfering with metadherin is likely to result in a decrease in tumorproliferation, especially when the interfering agent is co-administeredwith another anti-proliferative agent. Accordingly, in some embodiments,the present invention provides methods of treating cancer (e.g.,metastatic breast cancer and, in particular, poor prognosis breastcancer). In other embodiments, the present invention provides methods ofpreventing cancer metastasis (e.g., metastatic breast cancer).

A. Antibody Therapy

In some embodiments, the present invention provides antibodies thattarget tumors that express metadherin. Any suitable antibody (e.g.,monoclonal, polyclonal, or synthetic) may be utilized in the therapeuticmethods disclosed herein. In some embodiments, antibodies are antibodiesto human metadherin. In other embodiments, antibodies are to a mouse (orother animal) metadherin homolog (i.e., the variant of the gene foundnaturally in that species).

In preferred embodiments, the antibodies used for cancer therapy arehumanized antibodies. Methods for humanizing are well known in the art(See e.g., U.S. Pat. Nos. 6,180,370, 5,585,089, 6,054,297, and5,565,332; each of which is herein incorporated by reference) but anyantibody modified by any means that makes the antibody more amenable touse in himans than the unmodified version is understood herein to be ahumanized antibody.

In some embodiments, the therapeutic antibodies comprise an antibodygenerated against metadherin, wherein the antibody is conjugated to acytotoxic agent. In such embodiments, a tumor specific therapeutic agentis generated that does not target normal cells, thus reducing many ofthe detrimental side effects of traditional chemotherapy. For certainapplications, it is envisioned that the therapeutic agents will bepharmacologic agents that will serve as useful agents for conjugation toantibodies, particularly cytotoxic or other anticellular agents havingthe ability to kill or suppress the growth or cell division of cells.The present invention contemplates the use of any pharmacologic agentthat can be conjugated to an antibody, and delivered in active form.Exemplary anticellular agents include chemotherapeutic agents,radioisotopes that emit cell-damaging radiation, and cytotoxins. Thetherapeutic antibodies of the present invention may include a variety ofcytotoxic conjugated agents, including but not limited to, radioactiveisotopes (e.g., iodine-131, iodine-123, technicium-99m, indium-111,rhenium-188, rhenium-186, gallium-67, copper-67, yttrium-90, iodine-125or astatine-211), hormones such as a steroid, antimetabolites such ascytosines (e.g., arabinoside, fluorouracil, methotrexate or aminopterin;an anthracycline; mitomycin C), vinca alkaloids (e.g., demecolcine;etoposide; mithramycin), and antitumor alkylating agent such aschlorambucil or melphalan. Other embodiments may include agents such asa coagulant, a cytokine, growth factor, bacterial endotoxin or the lipidA moiety (i.e., a portion of a molecule that accounts for a function ofthe molecule) of bacterial endotoxin. For example, in some embodiments,therapeutic agents will include plant-, fungus- or bacteria-derivedtoxin, such as an A chain toxins, a ribosome inactivating protein,u-sarcin, aspergillin, restrictocin, a ribonuclease, diphtheria toxin orpseudomonas exotoxin, to mention just a few examples. In some preferredembodiments, deglycosylated ricin A chain is utilized.

In any event, it is proposed that agents such as these may, if desired,be conjugated to an antibody, preferably in a manner that will allow theagent to be directed to the desired site in or on a tumor or tumor cell,effectively presented to the site and, if necessary or beneficial, to betaken up (internalized) by the target and/or released from the antibodyto which it is conjugated. Known conjugation technology to achieve anyor all of these objectives are well-known in the art (See, e.g., Ghoseet al., Methods Enzymol., 93:280 [1983]).

For example, in some embodiments the present invention providesimmunotoxins targeting metadherin. Immunotoxins are conjugates of aspecific targeting agent, typically a tumor-directed antibody orfragment, with a cytotoxic agent, such as a toxin moiety. The targetingagent directs the toxin to, and thereby selectively kills, cellscarrying the targeted antigen. In some embodiments, therapeuticantibodies employ crosslinkers that provide high in vivo stability(Thorpe et al., Cancer Res., 48:6396 [1988]).

In preferred embodiments, antibody-based therapeutics are formulated aspharmaceutical compositions as described below. In preferredembodiments, administration of an antibody composition of the presentinvention results in a measurable decrease in cancer (e.g., decrease orelimination of tumor).

B. Antisense Therapies

In some embodiments, the present invention targets the expression ofmetadherin. For example, in some embodiments, the present inventionemploys compositions comprising oligomeric antisense compounds,particularly oligonucleotides (e.g., those identified in the drugscreening methods described herein), for use in modulating the functionof nucleic acid molecules encoding metadherin, ultimately modulating theamount of metadherin expressed. This is accomplished by providingantisense compounds that specifically hybridize with one or more nucleicacids encoding metadherin. The specific hybridization of an oligomericcompound with its target nucleic acid interferes with the normalfunction of the nucleic acid. This modulation of function of a targetnucleic acid by compounds that specifically hybridize to it is generallyreferred to as “antisense.” The functions of DNA to be interfered withinclude replication and transcription. The functions of RNA to beinterfered with include all vital functions such as, for example,splicing of the RNA to yield one or more mRNA species, translocation ofthe RNA from the nucleus to the site of protein translation in theendoplasmic reticulum, translation of protein from the RNA, andcatalytic activity that may be engaged in or facilitated by the RNA. Theoverall effect of such interference with target nucleic acid function ismodulation of the expression of metadherin. In the context of thepresent invention, “modulation” means either an increase (stimulation)or a decrease (inhibition) in the expression of a gene. For example,expression may be inhibited to potentially prevent tumor proliferation.

It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of the present invention, is a multistep process. The processusually begins with the identification of a nucleic acid sequence whosefunction is to be modulated. This may be, for example, a gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding metadherin. The targeting process also includesdetermining a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred site in the gene is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since the translation initiation codonis typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes).Eukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the present invention, “startcodon” and “translation initiation codon” refer to the codon or codonsthat are used in vivo to initiate translation of an mRNA moleculetranscribed from a gene encoding a tumor antigen of the presentinvention, regardless of the sequence(s) of such codons.

Translation termination codons (or “stop codon”) of a gene may have oneof three sequences (i.e., 5′-UAA, 5′-UAG and 5′-UGA; the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms“start codon region” and “translation initiation codon region” refer toa portion of such an mRNA or gene that encompasses from about 25 toabout 50 contiguous nucleotides in either direction (i.e., 5′ or 3′)from a translation initiation codon. Similarly, the terms “stop codonregion” and “translation termination codon region” refer to a portion ofsuch an mRNA or gene that encompasses from about 25 to about 50contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon.

The open reading frame (ORF) or “coding region,” which refers to theregion between the translation initiation codon and the translationtermination codon, is also a region that may be targeted effectively.Other target regions include the 5′ untranslated region (5′ UTR),referring to the portion of an mRNA in the 5′ direction from thetranslation initiation codon, and thus including nucleotides between the“5′ cap site” and the translation initiation codon of an mRNA orcorresponding nucleotides on the gene, and the 3′ untranslated region(3′ UTR), referring to the portion of an mRNA in the 3′ direction fromthe translation termination codon, and thus including nucleotidesbetween the translation termination codon and 3′ end of an mRNA orcorresponding nucleotides on the gene. The 5″ cap site of an mRNAcomprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap. The cap region may alsobe a preferred target region.

mRNA splice sites (i.e., intron-exon junctions) may also be preferredtarget regions, and are particularly useful in situations where aberrantsplicing is implicated in disease, or where an overproduction of aparticular mRNA splice product is implicated in disease. Aberrant fusionjunctions due to rearrangements or deletions are also preferred targets.It has also been found that introns can also be effective, and thereforepreferred, target regions for antisense compounds targeted, for example,to DNA or pre-mRNA.

In some embodiments, target sites for antisense inhibition areidentified using commercially available software programs (e.g.,Biognostik, Gottingen, Germany; SysArris Software, Bangalore, India;Antisense Research Group, University of Liverpool, Liverpool, England;GeneTrove, Carlsbad, Calif.). In other embodiments, target sites forantisense inhibition are identified using the accessible site methoddescribed in Patent WO0198537A2, herein incorporated by reference.

Once one or more target sites have been identified, oligonucleotides arechosen that are sufficiently complementary to the target (i.e.,hybridize sufficiently well and with sufficient specificity) to give thedesired effect. For example, in preferred embodiments of the presentinvention, antisense oligonucleotides are targeted to or near the startcodon.

In the context of this invention, “hybridization,” with respect toantisense compositions and methods, means hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases. For example, adenine andthymine are complementary nucleobases that pair through the formation ofhydrogen bonds. It is understood that the sequence of an antisensecompound need not be 100% complementary to that of its target nucleicacid to be specifically hybridizable. An antisense compound isspecifically hybridizable when binding of the compound to the target DNAor RNA molecule interferes with the normal function of the target DNA orRNA to cause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the antisense compoundto non-target sequences under conditions in which specific binding isdesired (i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed).

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with specificity, can be used to elucidate thefunction of particular genes. Antisense compounds are also used, forexample, to distinguish between functions of various members of abiological pathway.

The specificity and sensitivity of antisense is also applied fortherapeutic uses. For example, antisense oligonucleotides have beenemployed as therapeutic moieties in the treatment of disease states inanimals and man. Antisense oligonucleotides have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides areuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues, and animals,especially humans.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 30 bases (i.e., fromabout 8 to about 30 linked bases), although both longer and shortersequences may find use with the present invention. Particularlypreferred antisense compounds are antisense oligonucleotides, even morepreferably those comprising from about 12 to about 25 bases.

Specific examples of preferred antisense compounds useful with thepresent invention include oligonucleotides containing modified backbonesor non-natural internucleoside linkages. As defined in thisspecification, oligonucleotides having modified backbones include thosethat retain a phosphorus atom in the backbone and those that do not havea phosphorus atom in the backbone. For the purposes of thisspecification, modified oligonucleotides that do not have a phosphorusatom in their internucleoside backbone can also be considered to beoligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage (i.e., the backbone) of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science 254:1497 (1991).

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂, —NH—O—CH₂, —CH₂—N(CH₃)—O—CH₂—[knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH ₂—, and —O—N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as—O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m),CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH_(2 n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃ OCF₃,SOCH₃, SO₂CH₃ ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the properties of an oligonucleotide with respect to what theoligonucleotide does functionally (i.e., its “pharmacodynamic”properties), and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′—O—CH₂CH₂OCH₃, alsoknown as 2′—O—(2-methoxyethyl) or 2′-MOE) (Martin et al., Hely. Chim.Acta 78:486 [1995]) i.e., an alkoxyalkoxy group. A further preferredmodification includes 2′-dimethylaminooxyethoxy (i.e., a O(CH₂)₂ON(CH₃)₂group), also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (alsoknown in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other preferred modifications include 2′-methoxy(2′-O—CH₃),2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substitutedadenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808. Certainof these nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2. degree ° C. andare presently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Another modification of the oligonucleotides of the present inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates that enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety, cholic acid,a thioether, (e.g., hexyl-5-tritylthiol), a thiocholesterol, analiphatic chain, (e.g., dodecandiol or undecyl residues), aphospholipid, (e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or apolyethylene glycol chain or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

One skilled in the relevant art knows well how to generateoligonucleotides containing the above-described modifications. Thepresent invention is not limited to the antisense oligonucleotidesdescribed above. Any suitable modification or substitution may beutilized.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds that are chimeric compounds. “Chimeric”antisense compounds or antisense “chimeras,” in the context of thepresent invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, and/or increased bindingaffinity for the target nucleic acid. An additional region of theoligonucleotide may serve as a substrate for enzymes capable of cleavingRNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellularendonuclease that cleaves the RNA strand of an RNA:DNA duplex at aninternal site. Activation of RNase H, therefore, results in cleavage ofthe RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric antisense compounds of the present invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above.

The present invention also includes pharmaceutical compositions andformulations that include the antisense compounds of the presentinvention as described below.

C. RNA Interference (RNAi)

In other embodiments, RNAi is utilized to inhibit metadherin function.RNAi represents an evolutionary conserved cellular defense forcontrolling the expression of foreign genes in most eukaryotes,including humans. RNAi is typically triggered by double-stranded RNA(dsRNA) and causes sequence-specific mRNA degradation of single-strandedtarget RNAs homologous in response to dsRNA. The mediators of mRNAdegradation are small interfering RNA duplexes (siRNAs), which arenormally produced from long dsRNA by enzymatic cleavage in the cell.siRNAs are generally approximately twenty-one nucleotides in length(e.g. 21-23 nucleotides in length), and have a base-paired structurecharacterized by two nucleotide 3′-overhangs. Following the introductionof a small RNA, or RNAi, into the cell, it is believed the sequence isdelivered to an enzyme complex called RISC(RNA-induced silencingcomplex). RISC recognizes the target and cleaves it with anendonuclease. It is noted that if larger RNA sequences are delivered toa cell, RNase III enzyme (Dicer) converts longer dsRNA into 21-23 nt dssiRNA fragments.

The transfection of siRNAs into animal cells results in the potent,long-lasting post-transcriptional silencing of specific genes (Caplen etal, Proc Natl Acad Sci U.S.A. 2001; 98: 9742-7; Elbashir et al., Nature.2001; 411:494-8; Elbashir et al., Genes Dev. 2001; 15: 188-200; andElbashir et al., EMBO J. 2001; 20: 6877-88, all of which are hereinincorporated by reference). Methods and compositions for performing RNAiwith siRNAs are described, for example, in U.S. Pat. No. 6,506,559,herein incorporated by reference.

siRNAs are extraordinarily effective at lowering the amounts of targetedRNA, and by extension proteins, frequently to undetectable levels. Thesilencing effect can last several months, and is extraordinarilyspecific, because one nucleotide mismatch between the target RNA and thecentral region of the siRNA is frequently sufficient to preventsilencing (Brummelkamp et al, Science 2002; 296:550-3; and Holen et al,Nucleic Acids Res. 2002; 30:1757-66, both of which are hereinincorporated by reference).

An important factor in the design of siRNAs is the presence ofaccessible sites for siRNA binding. Bahoia et al., (J. Biol. Chem.,2003; 278: 15991-15997; herein incorporated by reference) describe theuse of a type of DNA array called a scanning array to find accessiblesites in mRNAs for designing effective siRNAs. These arrays compriseoligonucleotides ranging in size from monomers to a certain maximum,synthesised using a physical barrier (mask) by stepwise addition of eachbase in the sequence. Thus, the arrays represent a full oligonucleotidecomplement of a region of the target gene. Hybridisation of the targetmRNA to these arrays provides an exhaustive accessibility profile ofthis region of the target mRNA. Such data are useful in the design ofantisense oligonucleotides (ranging from 7mers to 25mers), where it isimportant to achieve a compromise between oligonucleotide length andbinding affinity, to retain efficacy and target specificity (Sohail etal, Nucleic Acids Res., 2001; 29(10): 2041-2045). Additional methods andconcerns for selecting siRNAs are described for example, in WO 05054270,WO05038054A1, WO03070966A2, J Mol. Biol. 2005 May 13; 348(4):883-93, JMol. Biol. 2005 May 13; 348(4):871-81, and Nucleic Acids Res. 2003 Aug.1; 31(15):4417-24, each of which is herein incorporated by reference inits entirety. In addition, software (e.g., the MWG online siMAX siRNAdesign tool) is commercially or publicly available for use in theselection of siRNAs.

D. Genetic Therapies

The present invention contemplates the use of any genetic manipulationfor use in modulating the expression of metadherin. Examples of geneticmanipulation include, but are not limited to, gene knockout (e.g.,removing the metadherin gene from the chromosome using, for example,recombination), expression of antisense constructs with or withoutinducible promoters, and the like. Delivery of nucleic acid construct tocells in vitro or in vivo may be conducted using any suitable method. Asuitable method is one that introduces the nucleic acid construct intothe cell such that the desired event occurs (e.g., expression of anantisense construct).

Introduction of molecules carrying genetic information into cells isachieved by any of various methods including, but not limited to,directed injection of naked DNA constructs, bombardment with goldparticles loaded with said constructs, and macromolecule-mediated genetransfer using, for example, liposomes, biopolymers, and the like.Preferred methods use gene delivery vehicles derived from viruses,including, but not limited to, adenoviruses, retroviruses, vacciniaviruses, and adeno-associated viruses. Because of the higher efficiencyas compared to retroviruses, vectors derived from adenoviruses are thepreferred gene delivery vehicles for transferring nucleic acid moleculesinto host cells in vivo. Adenoviral vectors have been shown to providevery efficient in vivo gene transfer into a variety of solid tumors inanimal models and into human solid tumor xenografts in immune-deficientmice. Examples of adenoviral vectors and methods for gene transfer aredescribed in PCT publications WO 00/12738 and WO 00/09675 and U.S. Pat.Nos. 6,033,908, 6,019,978, 6,001,557, 5,994,132, 5,994,128, 5,994,106,5,981,225, 5,885,808, 5,872,154, 5,830,730, and 5,824,544, each of whichis herein incorporated by reference in its entirety.

Vectors may be administered to a subject in a variety of ways. Forexample, in some embodiments of the present invention, vectors areadministered into tumors or tissue associated with tumors using directinjection. In other embodiments, administration is via the blood orlymphatic circulation (See e.g., PCT publication 99/02685 hereinincorporated by reference in its entirety). Exemplary dose levels ofadenoviral vector are preferably 10⁸ to 10¹¹ vector particles added tothe perfusate.

E. Small Molecules

In still further embodiments, the present invention provides drugs(e.g., small molecule drugs) that target metadherin activity. In someembodiments, small molecule drugs are identified using the drugscreening methods described below. In other embodiments, small moleculedrugs are described in WO 04/071460, WO 04/071499, WO 03/084993, WO03/075853, WO 05/021500, WO 05/021499, U.S. Applications 20040171552 and20040138171, WO 03/072599, WO 05/021498, WO 05/020899, WO 04/098516 andWO 04/098512, each of which is herein incorporated by reference in itsentirety.

F. Combination Therapy

In still further embodiments, one or more of the above describedtherapeutic agents are administered in combination. In some embodiments,a combination of a known chemotherapy agent (e.g., paclitaxel) and anantibody directed towards metadherin are utilized in the treatment ofbreast cancer. In certain embodiments, combination therapy (e.g., usinga metadherin antibody and a known chemotherapy agent) is initiallyutilized, followed by maintenance therapy with a single agent (e.g., anantibody directed toward metadherin).

In some embodiments, the compounds of the present invention are providedin combination with known cancer chemotherapy agents. The presentinvention is not limited to a particular chemotherapy agent.

Various classes of antineoplastic (e.g., anticancer) agents arecontemplated for use in certain embodiments of the present invention.Anticancer agents suitable for use with the present invention include,but are not limited to, agents that induce apoptosis, agents thatinhibit adenosine deaminase function, inhibit pyrimidine biosynthesis,inhibit purine ring biosynthesis, inhibit nucleotide interconversions,inhibit ribonucleotide reductase, inhibit thymidine monophosphate (TMP)synthesis, inhibit dihydrofolate reduction, inhibit DNA synthesis, formadducts with DNA, damage DNA, inhibit DNA repair, intercalate with DNA,deaminate asparagines, inhibit RNA synthesis, inhibit protein synthesisor stability, inhibit microtubule synthesis or function, and the like.

In some embodiments, exemplary anticancer agents suitable for use incompositions and methods of the present invention include, but are notlimited to: 1) alkaloids, including microtubule inhibitors (e.g.,vincristine, vinblastine, and vindesine, etc.), microtubule stabilizers(e.g., paclitaxel, and docetaxel, etc.), and chromatin functioninhibitors, including topoisomerase inhibitors, such asepipodophyllotoxins (e.g., etoposide (VP-16), and teniposide (VM-26),etc.), and agents that target topoisomerase I (e.g., camptothecin andisirinotecan (CPT-11), etc.); 2) covalent DNA-binding agents (alkylatingagents), including nitrogen mustards (e.g., mechlorethamine,chlorambucil, cyclophosphamide, ifosphamide, and busulfan, etc.),nitrosoureas (e.g., carmustine, lomustine, and semustine, etc.), andother alkylating agents (e.g., dacarbazine, hydroxymethylmelamine,thiotepa, and mitomycin, etc.); 3) noncovalent DNA-binding agents(antitumor antibiotics), including nucleic acid inhibitors (e.g.,dactinomycin (actinomycin D), etc.), anthracyclines (e.g., daunorubicin(daunomycin, and cerubidine), doxorubicin (adriamycin), and idarubicin(idamycin), etc.), anthracenediones (e.g., anthracycline analogues, suchas bleomycins, etc., and plicamycin (mithramycin), 4) antimetabolites,including antifolates (e.g., methotrexate), purine antimetabolites(e.g., 6-mercaptopurine, 6-thioguanine (6-TG), azathioprine, acyclovir,ganciclovir, chlorodeoxyadenosine, 2-chlorodeoxyadenosine (CdA), and2′-deoxycoformycin (pentostatin), etc.), pyrimidine antagonists (e.g.,fluoropyrimidines), 5-fluorouracil, 5-fluorodeoxyuridine (FdUrd), etc.),and cytosine arabinosides; 5) enzymes, including L-asparaginase, andhydroxyurea, etc.; 6) hormones, including glucocorticoids, antiestrogens(e.g., tamoxifen, etc.), nonsteroidal antiandrogens (e.g., flutamide,etc.), and aromatase inhibitors (e.g., anastrozole); 7) platinumcompounds (e.g., cisplatin and carboplatin, etc.); 8) monoclonalantibodies conjugated with anticancer drugs, toxins, and/orradionuclides, etc.; 9) biological response modifiers (e.g., interferons(e.g., IFN-a, etc.) and interleukins (e.g., IL-2, etc.); 10) adoptiveimmunotherapy; 11) hematopoietic growth factors; 12) agents that inducetumor cell differentiation (e.g., all-trans-retinoic acid, etc.); 13)gene therapy techniques; 14) antisense therapy techniques; 15) tumorvaccines; 16) therapies directed against tumor metastases (e.g.,batimastat, etc.); 17) angiogenesis inhibitors; 18) proteosomeinhibitors; 19) inhibitors of acetylation and/or methylation (e.g., HDACinhibitors); 20) modulators of NF kappa B; 21) inhibitors of cell cycleregulation (e.g., CDK inhibitors); 22) modulators of p53 proteinfunction; and 23) radiation.

Any oncolytic agent that is routinely used in a cancer therapy contextfinds use in the compositions and methods of the present invention. Forexample, the U.S. Food and Drug Administration maintains a formulary ofoncolytic agents approved for use in the United States. Internationalcounterpart agencies to the U.S.F.D.A. maintain similar formularies.Table 3 provides a list of exemplary antineoplastic agents approved foruse in the U.S. Those skilled in the art will appreciate that the“product labels” required on all U.S. approved chemotherapeuticsdescribe approved indications, dosing information, toxicity data, andthe like, for the exemplary agents.

H. Pharmaceutical Compositions

The present invention further provides pharmaceutical compositions(e.g., comprising the therapeutic compounds described above). Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product.

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (WO 97/30731), also enhancethe cellular uptake of oligonucleotides.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents that function by a non-antisense mechanism.Examples of such chemotherapeutic agents include, but are not limitedto, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin,bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). Anti-inflammatory drugs, including but arenot limited to nonsteroidal anti-inflammatory drugs and corticosteroids,and antiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. Other non-antisense chemotherapeutic agents are also withinthe scope of this invention. Two or more combined compounds may be usedtogether or sequentially.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, and the nature of the drugs or therapeutic agentsadministered, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient. Theadministering physician can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual agents (such as oligonucleotides),and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models or based on the examples describedherein. In general, dosage is from 0.01 μg to 100 g per kg of bodyweight, and may be given once or more daily, weekly, monthly or yearly.The treating physician can estimate repetition rates for dosing based onmeasured residence times and concentrations of the drug in bodily fluidsor tissues. Following successful treatment, it may be desirable to havethe subject undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the oligonucleotide or other treatment agent isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily or at longer intervals.

-   II. Markers for Cancer

The present invention further provides markers whose expression isspecifically altered in cancerous tissues (including but not limited tobreast cancer tissues) including poor prognosis tissues. Such markersfind use in the diagnosis and characterization of breast cancer. Forexample, in some embodiments, increased levels of metadherin in breastsamples serve as an indicator of the presence of cancer or the presenceof cancer that has metastasized or is likely to metastasize (e.g., tolung).

In some embodiments, the present invention provides methods fordetection of expression of metadherin. In preferred embodiments,expression is measured directly (e.g., at the RNA or protein level). Insome embodiments, a method for detecting expression of metadherin and adifferent method for determining the number of copies of metadheringenes are both used and the “marker” is effectively the integratedresult of applying the two methods. In some embodiments, expression isdetected in tissue samples (e.g., biopsy tissue). In other embodiments,expression is detected in bodily fluids (e.g., including but not limitedto, plasma, serum, whole blood, mucus, prostatic secretions, and urine).The present invention further provides panels and kits for the detectionof markers. In preferred embodiments, the presence of a cancer marker(e.g., metadherin) is used to provide a prognosis to a subject. Forexample, the detection of increased levels of expression of metadherinin breast samples, especially when due to an increase in copy number, isassociated with tumors that have metastasized. The information providedis also used to direct the course of treatment. For example, if asubject is found to have a marker indicative of a highly metastasizingtumor, additional therapies (e.g., hormonal or radiation therapies) canbe started at an earlier point when they are more likely to be effective(e.g., before metastasis).

A. Detection of RNA

In some preferred embodiments, detection of metadherin is detected bymeasuring the expression of corresponding mRNA in a tissue sample (e.g.,breast tissue). mRNA expression may be measured by any suitable method,including but not limited to, those disclosed below.

In some embodiments, RNA is detected by Northern blot analysis. Northernblot analysis involves the separation of RNA and hybridization of acomplementary labeled probe.

In still further embodiments, RNA (or corresponding cDNA) is detected byhybridization of the RNA to be detected (the “target” RNA) to anoligonucleotide probe. A variety of hybridization assays using a varietyof technologies for hybridization and detection are available. Forexample, in some embodiments, TaqMan assay (PE Biosystems, Foster City,Calif.; See e.g., U.S. Pat. Nos. 5,962,233 and 5,538,848, each of whichis herein incorporated by reference) is utilized. The assay is performedduring a PCR reaction. The TaqMan assay exploits the 5′-3′ exonucleaseactivity of the AMPLITAQ GOLD DNA polymerase. A probe consisting of anoligonucleotide with a 5′-reporter dye (e.g., a fluorescent dye) and a3′-quencher dye is included in the PCR reaction. During PCR, if theprobe is bound to its target, the 5′-3′ nucleolytic activity of theAMPLITAQ GOLD polymerase cleaves the probe between the reporter and thequencher dye. The separation of the reporter dye from the quencher dyeresults in an increase of fluorescence. The signal accumulates with eachcycle of PCR and can be monitored with a fluorimeter.

In yet other embodiments, reverse-transcriptase PCR (RT-PCR) is used todetect the expression of RNA. In RT-PCR, RNA is enzymatically convertedto complementary DNA or “cDNA” using a reverse transcriptase enzyme. ThecDNA is then used as a template for a PCR reaction. PCR products can bedetected by any suitable method, including but not limited to, gelelectrophoresis and staining with a DNA specific stain or hybridizationto a labeled probe. In some embodiments, the quantitative reversetranscriptase PCR with standardized mixtures of competitive templatesmethod described in U.S. Pat. Nos. 5,639,606, 5,643,765, and 5,876,978(each of which is herein incorporated by reference) is utilized.

B. Detection of Protein

In other embodiments, expressed metadherin is detected by measuring theexpression of the corresponding protein or polypeptide. Proteinexpression may be detected by any suitable method. In some embodiments,proteins are detected by immunohistochemistry. In other embodiments,proteins are detected by their binding to an antibody raised against theprotein. The generation of antibodies is described herein.

Antibody binding is detected by techniques known in the art (e.g.,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (e.g., usingcolloidal gold, enzyme or radioisotope labels, for example), Westernblots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays, etc.), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.)

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many methods are known in the art for detecting binding in animmunoassay and are within the scope of the present invention.

In some embodiments, an automated detection assay is utilized. Methodsfor the automation of immunoassays include those described in U.S. Pat.Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which isherein incorporated by reference. In some embodiments, the analysis andpresentation of results is also automated. For example, in someembodiments, software that generates a prognosis based on the presenceor absence of a series of proteins corresponding to metadherin isutilized.

In other embodiments, the immunoassay described in U.S. Pat. Nos.5,599,677 and 5,672,480; each of which is herein incorporated byreference.

C. Data Analysis

In some embodiments, a computer-based analysis program is used totranslate the raw data generated by the detection assay (e.g., thepresence, absence, or amount of metadherin genes or expression products)into data of predictive value for a clinician. The clinician can accessthe predictive data using any suitable means. Thus, in some preferredembodiments, the present invention provides the further benefit that theclinician, who is not likely to be trained in genetics or molecularbiology, need not understand the raw data. The data is presenteddirectly to the clinician in its most useful form. The clinician is thenable to immediately utilize the information in order to optimize thecare of the subject.

The present invention contemplates any method capable of receiving,processing, and transmitting the information to and from laboratoriesconducting the assays, information providers, medical personal, andsubjects. For example, in some embodiments of the present invention, asample (e.g., a biopsy or a serum or urine sample) is obtained from asubject and submitted to a profiling service (e.g., clinical lab at amedical facility, genomic profiling business, etc.), located in any partof the world (e.g., in a country different than the country where thesubject resides or where the information is ultimately used) to generateraw data. Where the sample comprises a tissue or other biologicalsample, the subject may visit a medical center to have the sampleobtained and sent to the profiling center, or subjects may collect thesample themselves (e.g., a urine sample) and directly send it to aprofiling center. Where the sample comprises previously determinedbiological information, the information may be directly sent to theprofiling service by the subject (e.g., an information card containingthe information may be scanned by a computer and the data transmitted toa computer of the profiling center using an electronic communicationsystems). Once received by the profiling service, the sample isprocessed and a profile is produced (i.e., expression data), specificfor the diagnostic or prognostic information desired for the subject.

The profile data are then prepared in a format suitable forinterpretation by a treating clinician. For example, rather thanproviding raw expression data, the prepared format may represent adiagnosis or risk assessment (e.g., likelihood of metastasis) for thesubject, along with recommendations for particular treatment options.The data may be displayed to the clinician by any suitable method. Forexample, in some embodiments, the profiling service generates a reportthat can be printed for the clinician (e.g., at the point of care) ordisplayed to the clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point ofcare or at a regional facility. The raw data is then sent to a centralprocessing facility for further analysis and/or to convert the raw datato information useful for a clinician or patient. The central processingfacility provides the advantage of privacy (all data is stored in acentral facility with uniform security protocols), speed, and uniformityof data analysis. The central processing facility can then control thefate of the data following treatment of the subject. For example, usingan electronic communication system, the central facility can providedata to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the datausing the electronic communication system. The subject may chose furtherintervention or counseling based on the results. In some embodiments,the data is used for research use. For example, the data may be used tofurther optimize the inclusion or elimination of markers as usefulindicators of a particular condition or stage of disease.

D. Kits

In yet other embodiments, the present invention provides kits for thedetection and characterization of breast cancer. In some embodiments,the kits contain antibodies specific for metadherin, in addition todetection reagents and buffers. In other embodiments, the kits containreagents specific for the detection of mRNA or cDNA (e.g.,oligonucleotide probes or primers). In preferred embodiments, the kitscontain all of the components necessary to perform a detection assay,including all controls, directions for performing assays, and anynecessary software for analysis and presentation of results.

E. In Vivo Imaging

In some embodiments, in vivo imaging techniques are used to visualizethe expression of metadherin in an animal (e.g., a human or non-humanmammal). For example, in some embodiments, metadherin is labeled using alabeled antibody specific for metadherin. A specifically bound andlabeled antibody can be detected in an individual using an in vivoimaging method, including, but not limited to, radionuclide imaging,positron emission tomography, computerized axial tomography, X-ray ormagnetic resonance imaging method, fluorescence detection, andchemiluminescent detection. Methods for generating antibodies tometadherin are described herein.

The in vivo imaging methods of the present invention are useful in thediagnosis of cancers that express metadherin (e.g., breast cancer). Invivo imaging is used to visualize the presence of a marker indicative ofthe cancer. Such techniques allow for diagnosis without the use of anunpleasant biopsy. The in vivo imaging methods of the present inventionare also useful for providing prognoses to cancer patients. For example,the presence of a marker indicative of cancers likely to metastasize canbe detected. The in vivo imaging methods of the present invention canfurther be used to detect metastatic cancers in other parts of the body.

In some embodiments, reagents (e.g., antibodies) specific for metadherinare fluorescently labeled. The labeled antibodies are introduced into asubject (e.g., orally or parenterally). Fluorescently labeled antibodiesare detected using any suitable method (e.g., using the apparatusdescribed in U.S. Pat. No. 6,198,107, herein incorporated by reference).In other embodiments, antibodies are radioactively labeled.

The use of antibodies for in vivo diagnosis is well known in the art.Sumerdon et al., (Nuc. Med. Biol 17:247-254 [1990] have described anoptimized antibody-chelator for the radioimmunoscintographic imaging oftumors using Indium-111 as the label. Griffin et al., (J Clin One9:631-640 [1991]) have described the use of this agent in detectingtumors in patients suspected of having recurrent colorectal cancer. Theuse of similar agents with paramagnetic ions as labels for magneticresonance imaging is known in the art (Lauffer, Magnetic Resonance inMedicine 22:339-342 [1991]). The label used will depend on the imagingmodality chosen. Radioactive labels such as Indium-111, Technetium-99m,or Iodine-131 can be used for planar scans or single photon emissioncomputed tomography (SPECT). Positron emitting labels such asFluorine-19 can also be used for positron emission tomography (PET). ForMRI, paramagnetic ions such as Gadolinium (III) or Manganese (II) can beused.

Radioactive metals with half-lives ranging from 1 hour to 3.5 days areavailable for conjugation to antibodies, such as scandium-47 (3.5 days)gallium-67 (2.8 days), gallium-68 (68 minutes), technetium-99m (6hours), and indium-111 (3.2 days), of which gallium-67, technetium-99m,and indium-111 are preferable for gamma camera imaging, gallium-68 ispreferable for positron emission tomography.

A useful method of labeling antibodies with such radiometals is by meansof a bifunctional chelating agent, such as diethylenetriaminepentaaceticacid (DTPA), as described, for example, by Khaw et al. (Science 209:295[1980]) for In-111 and Tc-99m, and by Scheinberg et al. (Science215:1511 [1982]). Other chelating agents may also be used, but the1-(p-carboxymethoxybenzyl)EDTA and the carboxycarbonic anhydride of DTPAare advantageous because their use permits conjugation without affectingthe antibody's immunoreactivity substantially.

Another method for coupling DPTA to proteins is by use of the cyclicanhydride of DTPA, as described by Hnatowich et al. (Int. J. Appl.Radiat. Isot. 33:327 [1982]) for labeling of albumin with In-111, butwhich can be adapted for labeling of antibodies. A suitable method oflabeling antibodies with Tc-99m which does not use chelation with DPTAis the pretinning method of Crockford et al., (U.S. Pat. No. 4,323,546,herein incorporated by reference).

A preferred method of labeling immunoglobulins with Tc-99m is thatdescribed by Wong et al. (Int. J. Appl. Radiat. Isot., 29:251 [1978])for plasma protein, and recently applied successfully by Wong et al. (J.Nucl. Med., 23:229 [1981]) for labeling antibodies.

In the case of the radiometals conjugated to the specific antibody, itis likewise desirable to introduce as high a proportion of theradiolabel as possible into the antibody molecule without destroying itsimmunospecificity. A further improvement may be achieved by effectingradiolabeling in the presence of metadherin, to insure that the antigenbinding site on the antibody will be protected. The antigen is separatedafter labeling.

In still further embodiments, in vivo biophotonic imaging is utilizedfor in vivo imaging. This real-time in vivo imaging utilizes luciferase.The luciferase gene is incorporated into cells, microorganisms, andanimals (e.g., as a fusion protein with METADHERIN). When active, itleads to a reaction that emits light. A CCD camera and software is usedto capture the image and analyze it.

-   III. Antibodies

The present invention provides antibodies having an affinity for (i.e.,a propensity to bind) peptides of interest herein, in particular,metadherin. In preferred embodiments, the present invention providesmonoclonal antibodies that specifically bind to a polypeptide comprisedof at least five amino acid residues (i.e., having at least five aminoacids, which may be identical or non-identical) . These find use in thediagnostic and therapeutic methods described herein.

Antibodies against a protein of the present invention may be monoclonalor polyclonal, as long as they can recognize the protein (as evidencedby binding to it). Antibodies can be produced by using a protein of thepresent invention as the antigen according to a conventional antibody orantiserum preparation process.

Any suitable method may be used to generate the antibodies used in themethods and compositions of the present invention, including but notlimited to, those disclosed herein. For example, to prepare eithermonoclonal or polyclonal antibodies, a protein as such or together witha suitable carrier or diluent is administered to an animal (e.g., amammal) under conditions that permit the production of antibodies. Thatis, the protein is to serve as an antigen. For enhancing the antibodyproduction capability, complete or incomplete Freund's adjuvant may beadministered. Normally, the protein (i.e., antigen) is administered onceevery 2 weeks to 6 weeks, in total, about 2 times to about 10 times.Animals suitable for use in such methods include, but are not limitedto, primates, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, etc.

For preparing monoclonal antibody-producing cells, an animal so treated(e.g., a mouse) is selected by “titrating” various dilutions of theanimal's blood serum against a given amount of the antigen. A serum—ofany dilution—that reacts with the antibody is an “antiserum.” The mostdilute antiserum that binds say, 50% of the antigen, is said to have thehighest titer. Two to 5 days after the final immunization, the selectedanimal's spleen or lymph node is harvested and antibody-producing cellscontained therein are isolated from one another, optionally alowed toundergo cell divisions thereafter, and then fused with myeloma cells toprepare the desired monoclonal antibody producer hybridoma. Measurementof the antibody titer in antiserum can be carried out, for example, byreacting a protein, labeled as described hereinafter, and antiserum andthen measuring the activity of the labeling agent bound to the antibody.The cell fusion can be carried out according to known methods, forexample, the method described by Koehler and Milstein (Nature 256:495[1975]). As a fusion promoter, for example, polyethylene glycol (PEG) orSendai virus (HVJ), preferably PEG is used.

Examples of myeloma cells include NS-1, P3U1, SP2/0, AP-1 and the like.The proportion of the number of antibody producer cells (spleen cells)and the number of myeloma cells to be used is preferably about 1:1 toabout 20:1. PEG (preferably PEG 1000-PEG 6000) is added in concentrationof about 10% to about 80%. Cell fusion can be carried out efficiently byincubating a mixture of both cells at about 20° C. to about 40° C.,preferably about 30° C. to about 37° C. for about 1 minute to 10minutes.

Various methods may be used to screen for hybridomas that are producingan antibody. For example, a supernatant of the hybridoma may be added toa solid phase (e.g., a microplate) that is capable of adsorbingantibody.Then an anti-immunoglobulin antibody (if mouse cells are usedin cell fusion, anti-mouse immunoglobulin antibody is used) or Protein A(a “universal” binder of immunoglobulins) labeled with a radioactivesubstance or an enzyme is added to detect the monoclonal antibodyagainst the protein bound to the solid phase. Alternately, a supernatantof the hybridoma is added to a solid phase to which ananti-immunoglobulin antibody or Protein A is adsorbed and then theprotein labeled with a radioactive substance or an enzyme is added todetect the monoclonal antibody against the protein bound to the solidphase.

Selection of the monoclonal antibody can be carried out according to anyknown method or its modification., A medium for animal cells to whichHAT (hypoxanthine, aminopterin, thymidine) is added may be employed. Anyselection and growth medium can be employed as long as the hybridoma cangrow. For example, RPMI 1640 medium containing 1% to 20% (preferably 10%to 20%) fetal bovine serum, GIT medium containing 1% to 10% fetal bovineserum, or a serum free medium for cultivation of a hybridoma (SFM-101,Nissui Seiyaku) and the like can be used. Normally, the cultivation iscarried out at 20° C. to 40° C.(preferably 37° C.) for about 5 days to 3weeks(preferably 1 week to 2 weeks) under air or oxygen containing about5% CO₂ gas. The antibody titer of the supernatant of a hybridoma culturecan be measured according to the same manner as described herein withrespect to the antibody titer of an antiserum.

Separation and purification of a monoclonal antibody (e.g., againstmetadherin) can be carried out according to any of the well-knownmethods for separation and purification of immunoglobulins, for example,salting-out, alcoholic precipitation, isoelectric point precipitation,electrophoresis, adsorption and desorption with ion exchangers (e.g.,DEAE), ultracentrifugation, gel filtration, or a specific purificationmethod wherein an antibody is collected with an active adsorbent such asan antigen-binding solid phase, Protein A or Protein G and dissociatingthe complex to obtain the antibody.

Polyclonal antibodies may be prepared by any of a number of well-knownmethods or modifications of these methods.. Briefly, an immunogen (i.e.,any agent capable of inducing an immune system to mount an immuneresponse when challenged by that agent) or a complex of an immunogen anda carrier (typically, a protein) is prepared and an animal is immunizedby the complex according to the same manner as that described above forpreparing a monoclonal antibody. As to the complex of the immunogen andthe carrier protein to be used for immunization of an animal, anycarrier protein and any mixing proportion of the carrier and a hapten(defined herein as an antigen to which the immune system respondsoptimally only if presented to the system with the carrier) can beemployed as long as an antibody against the hapten, which is crosslinkedon the carrier and used for immunization, is produced. For example,bovine serum albumin, bovine cycloglobulin, keyhole limpet hemocyanin,etc. may be coupled to an hapten in a weight ratio of about 0.1 part toabout 20 parts, preferably, about 1 part to about 5 parts per 1 part ofthe hapten.

In addition, various agents can be used for coupling (“condensing”) of ahapten and a carrier. For example, glutaraldehyde, carbodiimide,maleimide activated ester, activated ester reagents containing a thiolgroup or dithiopyridyl group, and the like find use with the presentinvention. The condensation product as such or together with a suitablecarrier or diluent is administered to a site of an animal that permitsof antibody production. For enhancing the antibody productioncapability, complete or incomplete Freund's adjuvant may beadministered. Normally, the protein is administered once every 2 weeksto 6 weeks, in total, about 3 times to about 10 times.

The polyclonal antibody is recovered from blood, ascites (peritonealfluid) and the like, of an animal immunized by the above method or froma subject who produces such antibodies as a result of having beenchallenged with an immunogen. The antibody titer in the antiserum can bemeasured in the manner described above with respect to the supernatantof the hybridoma culture. Separation and purification of the antibodycan be carried out as described above with respect to the monoclonalantibody. In alternative embodiments, polyclonal antibodies in ascitesfluid or in serum prepared from blood may be used without furtherisolation or purification. Such an antibody-containing serum isgenerally referred to as “antiserum.”

The protein used herein as the immunogen is not limited to anyparticular type of immunogen. For example, metadherin (including aprotein expression product of a metadherin gene having a partly alterednucleotide sequence) can be used as the immunogen. Further, fragments ofthe protein may be used. Fragments may be obtained by any methodsincluding, but not limited to expressing a fragment of the gene,enzymatic processing of the protein, chemical synthesis, and the like.

-   IV. Drug Screening

In some embodiments, the present invention provides drug screeningassays (e.g., to screen for anticancer drugs). In some embodiments, thescreening methods of the present invention utilize metadherin. Forexample, in some embodiments, the present invention provides methods ofscreening for compounds that alter (e.g., increase or decrease) theexpression of metadherin. In some embodiments, candidate compounds areantisense or siRNA agents (e.g., oligonucleotides) directed againstmetadherin. In other embodiments, candidate compounds are antibodiesthat specifically bind to metadherin. In yet other embodiments,candidate compounds are small molecules (i.e., biologically active butnon-polymeric) that inhibit a biological activity of metadherin.

In one screening method, candidate compounds are evaluated for theirability to alter metadherin expression by contacting a compound with acell expressing metadherin and then assaying for the effect of thecandidate compounds on expression. In some embodiments, the effect ofcandidate compounds on expression of metadherin is assayed for bydetecting the level of metadherin mRNA expressed by the cell. mRNAexpression can be detected by any suitable method.

In other embodiments, the effect of candidate compounds on expression ofa gene of interest (e.g., metadherin) is assayed by measuring the levelof expressed polypeptide. The level of polypeptide expressed can bemeasured using any suitable method, including but not limited to, thosedisclosed herein.

Specifically, the present invention provides screening methods foridentifying modulators, i.e., candidate or test compounds or agents(e.g., proteins, peptides, peptidomimetics, peptoids, small molecules orother drugs) which bind to metadherin, have an inhibitory effect on, forexample, metadherin expression or metadherin activity, or have astimulatory or inhibitory effect on, for example, the expression oractivity of a metadherin substrate. Compounds thus identified can beused to modulate the activity of metadherin or other target gene producteither directly or indirectly in a therapeutic protocol, to elaboratethe biological function of the target gene product, or to identifycompounds that disrupt normal target gene interactions. Compounds whichinhibit the activity or expression of metadherin are useful in thetreatment of proliferative disorders, e.g., cancer, particularlymetastatic (e.g., to the lung) breast cancer.

In one embodiment, the invention provides assays for screening candidateor test compounds that are substrates of metadherin protein orpolypeptide or a biologically active portion thereof. In anotherembodiment, the invention provides assays for screening candidate ortest compounds that bind to or modulate the activity of metadherinprotein or polypeptide or a biologically active portion thereof.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including biological libraries (e.g., micro-organisms); peptoidlibraries (libraries of molecules having the functionalities ofpeptides, but with a novel, non-peptide backbone, which are resistant toenzymatic degradation but which nevertheless remain bioactive; see,e.g., Zuckennann et al., J., Med. Chem. 37: 2678-85 [1994]); spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution (a mathematical means ofmanaging “noise” in bodies of data); the ‘one-bead one-compound’ librarymethod; and synthetic library methods using affinity chromatographyselection. The biological library and peptoid library approaches arepreferred for use with peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer (as usedherein, a polymer having only a few residues or “mers”) or smallmolecule libraries of compounds (Lam (1997) Anticancer Drug Des.12:145). The term “library” herein means a collection of one or anotherof the aforementnioned classes of agents or the like, assembledpreferably according to a theme or algorithm constructed according toprinciples of combinatorial mathematics such as game theory—thus theterm “combinatorial library.”

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90:6909 [1993]; Erb et al., Proc. Nad. Acad. Sci. USA 91:11422[1994]; Zuckermann et al., J. Med. Chem. 37:2678 [1994]; Cho et al.,Science 261:1303 [1993]; Carrell et al., Angew. Chem. Int. Ed. Engl.33.2059 [1994]; Care11 et al., Angew. Chem. Int. Ed. Engl. 33:2061[1994]; and Gallop et al., J. Med. Chem. 37:1233 [1994].

Compounds selected from libraries may be presented in solution (e.g.,Houghten, Biotechniques 13:412-421 [1992]), or in or on beads (Lam,Nature 354:82-84 [1991]), chips (Fodor, Nature 364:555-556 [1993]),bacteria or spores (U.S. Pat. No. 5,223,409; herein incorporated byreference), plasmids (Cull et al., Proc. Nad. Acad. Sci. USA 89:18651869[1992]) or phage (Scott and Smith, Science 249:386-390 [1990]; DevlinScience 249:404-406 [1990]; Cwirla et al., Proc. Natl. Acad. Sci.87:6378-6382 [1990]; Felici, J. Mol. Biol. 222:301 [1991]).

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein(e.g., a metadherin modulating agent, an antisense metadherin nucleicacid molecule, a siRNA molecule, a metadherin-specific antibody, or ametadherin-binding partner) in an appropriate animal model (such asthose described herein) to determine the efficacy, toxicity orsideeffects of treatment with such an agent, or to elucidate its mechanismof action. Furthermore, novel agents identified by the above-describedscreening assays can be, e.g., used for treatments as described herein.

-   V. Transgenic Animals Expressing Metadherin Genes

The present invention contemplates the generation of transgenic animalsthat over-express or under-express (e.g., knockout animals) metadherin.The transgenic animals of the present invention find use in drug (e.g.,cancer therapy) screens. In some embodiments, test compounds (e.g., adrug that is suspected of being useful to treat cancer) and controlcompounds (e.g., a placebo) are administered to the transgenic animalsand the control animals and the effects evaluated..

EXPERIMENTAL

The following are examples that further illustrate embodimentscontemplated by the present invention. It is not intended that theseexamples provide any limitations on the present invention.

In the experimental disclosure that follows, the following abbreviationsapply: eq.

-   or eqs. (equivalents); M (Molar); .mu.M (micromolar); N (Normal);    mol (moles); mmol-   (millimoles); .mu.mol (micromoles); nmol (nanomoles); pmoles    (picomoles); g (grams);-   mg (milligrams); .mu.g (micrograms); ng (nanogram); vol (volume);    w/v (weight to volume); v/v (volume to volume); L (liters); ml    (milliliters);.-   mu.1 (microliters); cm-   (centimeters); mm (millimeters); .mu.m (micrometers); nm    (nanometers); C (degrees-   Centigrade); rpm (revolutions per minute); DNA (deoxyribonucleic    acid); kdal (kilodaltons).-   I. Results

1. Recurrent Poor-Prognosis Genomic Alterations

A bioinformatic strategy termed ACE (Analysis of CNAs by Expressiondata) (FIG. 1a ) was developed to sensitively detect CNAs that affectregional gene expression. ACE first calculates the expression scores ofall genes according to expression differences between comparison groups,and then orders these scores based on genomic position. To measure theregional expression pattern, a neighborhood score (NS) is calculated foreach genomic locus using a geometry-weighted sum of expression scores ofall the genes on the chromosome. Since locus linkage strength decayswith distance, the expression scores of genes in proximity to the locusin consideration are assigned greater weights than those farther away.NS significance is estimated by permutation, with regions with a stretch(>20) of aberrant NS declared potential CNA regions.

Once the efficacy of the ACE method was validated using a number ofexisting gene expression profiling datasets that have correspondinggenomic alteration information (See FIG. 2), the method was used tostudy genomic alterations associated with poor prognosis human cancers,and in particular poor prognosis breast cancer. Three separate studiespreviously identified two poor-prognosis gene sets (70 and 76 genes,respectively) that can be used to robustly predict the clinical outcomeof human breast cancers. However, only a single gene (CCNE2) is presentin both signatures. Analysis of these three datasets using the ACEmethod identified five common genomic gains in at least two datasets(Table 1) and 15 other genomic gains in one of the three datasets(Supplementary Table 2). The smallest regions of overlap (SRO) of commonCNA events, namely, gains at 3q26-27, 8q22, 8q24.3, 17q23-25 and20q13.3, are among a large number of genomic alterations previouslyobserved in high frequencies in breast cancer, although their links topoor prognosis and tumor progression have not been established³⁵⁻⁴⁰.Genomic losses associated with more than one dataset were not detected.This is consistent with previous observations that genomic gains aremore prevalent than genomic losses in cancer, particularly poorprognosis breast cancer³⁸⁻⁴⁰. Of the five prevalent genomic events, the8q22 gain was consistently observed in all three datasets (FIG. 1b ).The NS of the 8q22 region was calculated for each sample in the threedatasets; the resulting score was used to classify tumor samples intotwo groups (high NS and low NS). FIG. 1c and Table 3, demonstrate thatthe probability of metastasis-free survival of patients with a high 8q22NS was significantly lower than the control group in all three datasets.These analyses suggested that the genomic gain of 8q22 is a strongpredictor of breast cancer poor prognosis.

a) FIG. 1

FIG. 1 demonstrates the use of ACE analysis to identify a recurrentgenomic gain at 8q22 in poor-prognosis breast cancer. FIG. 1a representsa schematic overview of the ACE approach. Briefly, the expression score(ES) of each gene is calculated by comparing samples of differentphenotypes, and then a neighborhood score (NS), indicative of the DNAamplification status, is computed for each locus as thegeometry-weighted ES sum of all the genes on the chromosome. Regions ofgain (red, bottom panel) and loss (green) were defined by applying NScutoffs (dotted lines) obtained from permutations. i,j, gene index whenthey are ordered on the chromosome by genomic positions; c, normalizingconstant; w_(ji), weight of gene j when locus i is in consideration.ES_(i), expression score of gene i. NS_(i), neighborhood score of locusi. FIG. 1b depicts the detection of a poor-prognosis genomic gain at8q22 in all three expression datasets by van't Veer¹⁴, van de Vijver¹⁵and Wang¹⁶ et al. The traces are the NS scores on chromosome 8 producedby ACE. The shaded area highlights the consensus region of gain at 8q22.Red and green peaks represent statistically significant regions of gainsor loss, respectively. FIG. 1c depicts Kaplan-Meier metastasis-freesurvival curves of patients with high or low 8q22 NS.

b) FIG. 2

FIG. 2 depicts the validation of the ACE algorithm using availableexpression data with corresponding genomic alteration data. FIG. 2adepicts expression microarray data of the brain tissue from Ts1Cje micecompared to that of the normal mice⁷¹ (Ts1Cje mice represent the DownSyndrome animal model known to have a partial trisomic region onchromosome 16). ACE predicted a sole CNA region on chromosome 16. The NSproduced by ACE is shown along this chromosome, where the red lineindicates the predicted region of gain. Red double-arrow: the knowntrisomic region in Ts1Cje mice. FIG. 2b depicts taxane-resistant cellsestablished by continuous exposure of docetaxel or paclitaxel to 6ovarian cancer cell lines 1A9, ES-2, MESOV, OVCA429, OVCA433 andOVCAR-3⁷². ACE detected 3 amplified regions on chromosome 7 in thetaxane-resistant derivatives when compared to their parental lines(upper panel), which were highly consistent with the analysis of CGHdata (lower panel). Colored horizontal lines in the lower panel are thesegment means produced by the CGH analysis tool CBS, of which redindicates the significant regions of gain. FIG. 2c demonstrates the useof ACE to compare the expression of 10 cell lines derived from thebreast cancer cell MDA-MB-231 with high or low breast-to-bone metastaticcapability⁹ and defined a loss at chromosome 7q associated with bonemetastasis. The upper panel shows the NS of chromosome 7 in the highlymetastatic cells with green lines indicating predicted regions of loss.The lower panel displays the previously published CGH data of the samechromosome in the two highly metastatic lines 2287 and 1833, with DNA ofthe lowly metastatic parental line MDA-MB-231 used as a control. Red andgreen vertical bars indicate regions of genomic loss and gain,respectively. FIG. 2d depicts the use of ACE to analyze regionalepigenetic regulation using the gene expression data of bladdertumors⁷⁰. Partial chromosome 3 is shown. Dark green double-arrow: theepigenetic regulated region that was experimentally validated in aprevious study ⁷⁰. See Table 4 for all significant regions in thisdataset.

2. Validation of 8₈22 Genomic Gain in Breast Tumors

Fluorescence in situ hybridization (FISH) and genomic DNA real-time PCR(qPCR) was used to confirm 8q22 amplification in breast tumor samples. Apanel of microdissected tumor samples from fresh frozen breast cancerspecimens was first analyzed by qPCR using four primer pairs thatamplify DNA sequences at chromosome 8q21, q22 and q23 (FIG. 3a , b). Asshown in Table 5, ten of 36 tumors (27.8%) were found to have aberrantlyhigher copy numbers (>3.6) at 8q22 as compared to control human DNAsample. As shown in FIG. 5b , these 10 genomic gain events spannedchromosomal regions 8q21 to 8q23, with a consensus region at 8q22. Thisresult is consistent with the computational prediction. DNA copy numbersdetected by genomic qPCR analysis are consistent with FISH analysis ofthe same tumor specimens (FIG. 7). To confirm the link between 8q22genomic gain and elevated expression of genes located in this region,qRT-PCR was used to investigate expression patterns of three genes at8q22 (PTDSSJ, MTDH and LAPTM4β) in these tumors. The resultsdemonstrated a strong positive correlation between the expression ofthese genes and the 8q22 copy numbers (FIG. 3b ). Analysis of a separatepanel of 18 paraffin-embedded breast tumors showed yielded similarresults (Supplementary Table 5).

A breast cancer tissue microarray with corresponding detailedclinicopathological records was also analyzed by FISH using a bacterialartificial chromosome (BAC) probe located at the 8q22 region. Resultsshowed that 22 (26.8%) of the 82 hybridized primary tumor samples had anaverage 8q22 copy number larger than 3 (FIG. 3c , Table 6). Notably,8q22 amplification was associated with a higher propensity of cancerrecurrence (FIG. 3d ). For example, about 65% of the patients with 8q22genomic gain suffered from metastasis 17 years after the initial cancerdiagnosis, as compared to 30% of patients without 8q22 amplification(Log rank P=0.002). Along with the qPCR analysis described above, thesedata confirmed the ACE prediction that recurrent genomic amplificationat 8q22 leads to regional gene activation. More importantly, theseresults established 8q22 amplification as a breast cancer poor-prognosismarker event.

a) FIG. 3

FIG. 3 depicts the validation of 8q22 amplification in human breasttumors. FIG. 3a demonstrates that the majority of the genes at the 8q22region are overexpressed in poor-prognosis tumor samples of the threepublished datasets¹⁴⁻¹⁶. Heatmap shows the differential expression ofthese genes in poor-prognosis vs. good-prognosis samples. Red indicatesoverexpression, while green denotes underexpression. FIG. 3b depicts thevalidation of the computational prediction of 8q22 genomic gain. A panelof human breast tumor samples obtained from LCM was analyzed for 8q22genomic alterations and gene expression using qPCR. Shown are the DNAcopy numbers of 4 genomic loci at 8q21-23 (filled circles) analyzed withthe extracted tumor DNA, and the expression levels of 3 genes at 8q22(diamonds) quantified with the tumor RNA. Student's t-test P values ofexpression comparison in samples with and without 8q22 gain are shown inparenthesis after each gene. FIG. 3c depicts breast cancer tissuemicroarray FISH analysis with the green SpectrumGreen and redSpectrumOrange probes detecting chromosome 8 centromere and the 8q22region, respectively. About 50 nuclei were scored per sample. A case of8q22 amplification (left) and a diploid case (right) were shown. FIG. 3ddepicts Kaplan-Meier survival analysis in breast cancer patients with orwithout 8q22 amplification.

b) FIG. 4

FIG. 4 depicts DNA copy number quantification by FISH and genomic DNAqPCR. Shown are FISH images of 2 paraffin tissue samples with red andgreen probes for 8q22 and chromosome 8 centromere, respectively. Theaverage 8q22 copy numbers scored from at least 100 nuclei in FISH andfrom the genomic qPCR assay were also shown for each sample.

3. MTDH Promotes Breast Cancer Metastasis

Thirteen of the 20 genes in 8q22 were represented on the microarraysused in the three analyzed datasets (FIG. 3a ). To determine thefunctional targets of 8q22 gain, six resident genes considered mostlikely to promote cancer progression were tested. UQCRB, PTDSS1, TSPYL5,MTDH and LAPTM4b were significantly overexpressed in metastatic diseasesin at least two of these datasets (student's t-test, P<0.05), and SDC2was reported to mediate cell adhesion and proliferation in coloncancer⁴¹ (FIG. 6). To examine their role in metastasis, each gene wasstably overexpressed in the SCP28 cell line, a subline of the humanbreast cell line MDA-MB-231 that is mildly metastatic to lung and bonewhen injected into mice^(9,42). The cell line was labeled with aretroviral construct expressing a GFP/luciferase fusion protein⁴², andits in vivo metastasis capability was monitored by non-invasivebioluminescent imaging after intravenous injection. Data showed thatMTDH overexpression significantly accelerated the development of lungmetastasis and shortened the survival of mice that received tumor cellxenografts (FIG. 5a-d and FIG. 6). Animal metastasis burden caused byMTDH overexpression was nearly 7-fold higher than controls six weeksafter cancer cell injection. In contrast, overexpression of the otherfive genes, either individually or in combination, failed to enhance themetastasis ability of SCP28 (FIG. 6), suggesting that MTDH is likely themost significant functional mediator of this poor-prognosis genomicgain. MTDH is located at the center of the 8q22 minimal consensusgenomic gain and has been shown to encode a cell surface proteinresponsible for promoting mouse mammary tumor cell adhesion to lungendothelial cells³². However, the functional role of MTDH in humanbreast cancer and the mechanism of its deregulation have not beenpreviously investigated. To further validate the role of MTDH inmetastasis, two different short-hairpin RNA (shRNA) constructs were usedto knock down the expression of MTDH in the LM2 cell line (an MDA-MB-231subline selected in vivo for its high lung metastasis propensity)¹¹.MTDH knockdown significantly reduced the lung metastasis burden of LM2by 3-5 fold and extended the survival of the mice by 1-2 weeks (FIGS. 5a, e-g and FIG. 6). The effect of altered MTDH expression on bone andbrain metastasis was also examined by injecting the genetically modifiedbreast cancer cell lines into the left cardiac ventricle of recipientnude mice. MTDH knockdown in LM2 resulted in a modest but significantimprovement of post-injection survival, although bioluminescentquantification of the decrease of bone and brain metastasis burden didnot reach statistical significance. Conversely, overexpression of MTDHin SCP28 cells led to a modest but significant increase of bone andbrain metastasis (FIG. 7). These results suggested that MTDHpreferentially promotes metastasis to lung, while having a modest effecton metastasis to other organs.

The functional role of MTDH in the multistep process of metastasis wasalso investigated^(4,5). MTDH knockdown or overexpression did not affectthe growth, migration or invasiveness of tumor cells (FIG. 8). However,MTDH knockdown significantly reduced the adhesion of the cancer cells tolung microvascular endothelial cells (HMVEC-L), as well as toendothelial cells of the bone marrow (HBMEC60) and the umbilical vein(HUVEC), albeit to a lesser extent. A reciprocal change was observedwhen MTDH was overexpressed (FIG. 5h ). In contrast, the adhesion ofcancer cells to the WI-38 lung fibroblast cell line was not affected.MTDH did not promote intravasation or extravasation through endotheliallayers based on both in vitro transendothelial assays (FIG. 8) and invivo metastasis assays using an orthotopic xenograft method (data notshown). Instead, MTDH appeared to specifically enhance the seeding oftumor cells to the target organ endothelium.

a) FIG. 5

FIG. 5 demonstrates that MTDH mediates lung metastasis of human breastcancer. FIG. 5a shows that MTDH is constitutively overexpressed in themildly metastatic cells SCP28, and stably knocked down in the highlylung-metastatic cells LM2 with two independent hairpin constructs. Shownare the Northern and Western blot results. FIG. 5b depicts an in vivometastasis assay of SCP28 cells with or without MTDH overexpression.Luciferase-labeled SCP28 cells were inoculated into nude miceintravenously, and the lung metastasis burden of xenografted animals wasmonitored weekly using non-invasive bioluminescent imaging (BLI). Shownare BLI images of representative mice at the sixth week after injection.The color scale depicts the photon flux (photon per second) emitted fromthe metastasis cells. FIG. 5c depicts BLI quantification of lungmetastasis of SCP28 cells. FIG. 5d depicts Kaplan-Meier survival curvesof mice inoculated with SCP28 cells. FIG. 5e depicts in vivo metastasisassays of LM2 cells with or without MTDH knockdown. Shown are therepresentative BLI images and lung sections of the inoculated mice atthe sixth week after injection. Arrows point to the sporadic lesions byMTDH knockdown cells as compared to much more prevalent tumor lesions bycontrol cells. FIG. 5f depicts BLI quantification of lung metastasis byLM2 cells. FIG. 5g depicts Kaplan-Meier survival analysis of the miceinjected with LM2 cells. FIGS. 5c and 5f data represent averages ±SEM of10 mice. *P<0.05; **P<0.01 based on a two-sided Wilcoxon rank test. FIG.5h demonstrates that MTDH promotes the adhesion of cancer cells toendothelial cells as tested by endothelial-adhesion assays. Geneticallymodified SCP28 or LM2 cells were seeded on top of a monolayer ofendothelial cells from lung (HMVEC-L), umbilical vein (HUVEL), bonemarrow (HBMEC60) and control fibroblast cells (WI38). Cancer cells wereseeded on top of the endothelial or fibroblast monolayer and theattached cells were quantified 3 hours later.

b) FIG. 6

FIG. 6 demonstrates the use overexpression analysis of 8q22 genes toidentify MTDH as the target gene of the amplicon to promote metastasis.Supplementary FIG. 6a depicts differential expression patterns of genesat 8q22 in patients with poor prognosis compared to those with goodprognosis. To identify the amplification target gene(s) among those, sixputative candidates including MTDH (color highlighted) with theexpression pattern most strongly correlated with prognosis or previouslyimplicated in tumor biology were chosen and their possible roles topromote metastasis were analyzed using the xenografting animal model.FIG. 6b depicts the analysis of SCP28 cells; cells that overexpress eachof the six genes as well as the empty vector were tested for theirmetastatic capability. The cells were injected into nude miceintravenously, followed by bioluminescent imaging to monitor the animallung metastasis burden. Shown are the normalized luminescent signalsfrom the cancer cells colonized in lung. Only MTDH overexpression led tosignificant increase of lung metastasis. *P<0.05; **P<0.01, two-sidedWilcoxon rank test to compare MTDH overexpression vs. control. FIG. 6cis included to rule out the possibility of a combinatory effect of theother genes by simultaneous overexpression in the SCP28 cells. FIG. 6ddemonstrates that xengrafting assays of the cells with combinationaloverexpressed did not show an increase of lung metastasis. FIG. 6edepicts photographs and hematoxylin/eosin stain sections ofrepresentative lungs harvested at necropsy from mice injected withcontrol and MTDH-knockdown LM2 cells.

c) FIG. 7

FIG. 7 demonstrates that MTDH mediates organ-specific metastasis. WhileMTDH shows a strong causal role in breast-to-lung metastasis, it onlymildly promotes breast-to-bone metastasis in mice. When the LM2 cellswith MTDH knockdown were inoculated via intracardiac injection into thenude mice to generate bone and brain metastasis, a slight decrease inthe bone metastasis (FIG. 7a ) and a modest but significant improvementof animal survival (FIG. 7b ) was observed. n=10. Reciprocally, MTDHoverexpression in SCP28 led to a significant increase in the bonemetastasis propensity (FIG. 7c ). n=10. *P<0.05 based on a two-sidedWilcoxon rank test. FIG. 7d depicts representative BLI images ofsystemic metastasis burden in mice injected with SCP28 control andMTDH-overexpression cells.

d) FIG. 8

FIG. 8 demonstrates that MTDH does not influence the growth, migrationor invasion of tumor cells. Supplementary FIG. 8a depicts LM2 cells withMTDH knockdown or control hairpin expression were inoculated into the #4mammary fat pad of nude mice. Length and width of the primary tumorswere measured, and the tumor volumes were calculated at the indicatedtime points. FIG. 8b demonstrates that the in vitro proliferation ratesof LM2 cells were not affected by MTDH knockdown. FIG. 8c depicts thegrowth curve of the SCP28 control or MTDH overexpression cells afterinoculation into mammary fat pads. FIG. 8d depicts the in vitroproliferation rates of SCP28 cells. Alteration of MTDH expression in LM2or SCP28 cells did not lead to change of migration and invasionproperties of the cancer cells as measured by wound healing assays (FIG.8e ), Boyden two-chamber migration assay (FIG. 8f ), and two-chambermatrigel invasion assay (FIG. 8g ). Results represent average values ofthree or more independent experiments with SEM as error bars.

4. MTDH Promotes Chemoresistance

Poor prognosis of breast cancer at the time of diagnosis or surgeryindicates a higher probability of death as the result of recurrenttumors and development of metastases in vital organs. Emergence ofmetastasis reflects not only the ability of cancer cells to overcomehurdles during the multi-step process of metastasis^(4,5), but also thecapability to survive standard adjuvant therapy and other physiologicalstresses. Therefore, the driver gene of a poor-prognosis geneticamplification might function to promote chemoresistance in addition toenabling the metastasis process. A bioinformatic analysis of theavailable NCl60 pharmacogenomic data⁴³ indicated a potentialcontribution of the genes at 8q22 to chemoresistance. The NCI60 datainclude the cytogenetic and expression profiles of 58 cancer cell linesas well as their sensitivity profiles to 24,000 small moleculecompounds. Analysis of such data revealed that genomic gain at 8q22strongly correlates with a higher overall gene expression of this region(Pearson's r=0.578, FIG. 10); intriguingly, this higher NS is in turnassociated with a significantly higher mean GI₅₀ (the drug concentrationfor 50% growth inhibition) for 1,123 compounds, as compared to 211±178compounds expected by random permutation (P=0.019, FIG. 9a ).

To investigate the chemoresistance function of MTDH and other genes in8q22, genetically modified breast cancer cell lines used for in vivometastasis assays were treated with chemotherapeutic or other stressagents including paclitaxel, doxorubicin, cisplatin, and hydrogenperoxide with or without co-culture with the HMVEC-L endothelial cellline. Long-term survival of the cells was then quantified by clonogenicassays. Inhibition of MTDH expression sensitized the LM2 cell line tochemotherapeutic and stress agents, while overexpression of MTDHrendered SCP28 cells more resistant to these treatments (FIG. 9b-d ). Incontrast, overexpression of up to 4 other genes in the 8q22 locus didnot significantly alter the chemosensitivity of cancer cells (FIG. 9d ).MTDH-dependent chemoresistance was further enhanced when cancer cellswere co-cultured with HMVEC-L lung endothelial cells (FIG. 9b, c ).

The chemoresistance function of MTDH was then examined in vivo usingxenograft models. LM2 cells with or without MTDH knockdown were injectedto nude mice subcutaneously. Twice-weekly treatment of tumors withpaclitaxel or the drug vehicle was initiated at one week afterinjection. Subcutaneous tumor volumes were monitored by direct calipermeasurement. When the mice were treated with the drug vehicle, the LM2tumors grew rapidly, reaching five times the initial volume in 18 daysafter treatment (FIG. 9e ). Tumors from the MTDH knockdown cells grew atan equal rate, an observation consistent with the finding that MTDH doesnot affect primary tumor growth (FIG. 8). Paclitaxel treatmentsignificantly hampered tumor growth in mice injected with the controlLM2 cells. However, the tumors still grew to 140% in volume 18 daysafter treatment, indicating a considerable degree of chemoresistance ofthese cancer cells. MTDH knockdown significantly sensitized the cells topaclitaxel treatment as tumor regression was observed immediately afterthe first treatment. The tumors eventually shrank to about 30% of thepre-treatment sizes 18 days after the initiation of treatment (FIG. 9e,f ). Similar results were obtained with another commonly usedchemotherapeutic agent doxorubicin (FIG. 11).

a) FIG. 9

FIG. 9 demonstrates that MTDH enhances chemoresistance of the breastcancer cells. FIG. 9a demonstrates that Genomic gain of 8q22 isassociated with higher resistance to chemical compounds in the 58 humancancer cell lines. logGI₅₀ (drug concentration for 50% growthinhibition) of each of the 24,642 compounds in cell lines with 8q22 gainwas compared to those in cells without 8q22 gain. The numbers ofcompounds with significantly increased logGI₅₀ in cells of 8q22 gain,counted by applying various significance thresholds of the logGI₅₀differences (P<0.05, 0.01 and 0.001), was compared to a nulldistribution obtained by permuting the 8q22 copy numbers of the celllines. Median values from permutations are shown with mean absolutedeviation (MAD) as the error bar. FIG. 9b demonstrates the analysis ofchemoresistance of LM2 cells using clonogenic assays after treatmentwith various apoptosis-inducing agents with or without HMVEC-Lco-culture. Shown are the relative clonogenic abilities as percentagesof the non-treatment control. FIG. 9c depicts representative images ofthe clonogenic assays for LM2 cells with or without MTDH-knockdown andHMVEC-L co-culture. FIG. 9d depicts clonogenic assays of SCP28 cellswith overexpression of MTDH or other genes in the amplicon. Shown arethe data with HMVEC-L co-culture. Results for FIGS. 9b and 9d representaverage values ±SEM of at least three independent experiments. In vivochemoresistance assay of LM2 cells with or without MTDH knockdown. Shownare the xenograft tumor sizes when mice were treated with Paclitaxel ordrug vehicle. 12 tumors per group were used. FIG. 9e depictsrepresentative tumors isolated from the mice 25 days after injection inthe in vivo chemoresistance assay. FIG. 9 b, d, e *P<0.05; **P<0.01;***P<0.001 with a two-sided student's t-test.

b) FIG. 10

FIG. 10 demonstrates the Correlation of 8q22 copy number in NS and NCI60cell lines. 8q22 DNA copy numbers are positively correlated with thegene expression levels of this region in the 58 human cancer cell linesof the NCI60 data. The 8q22 copy numbers were analyzed from SNPmicroarray data using CBS algorithm and shown as the segment meanvalues. The overall 8q22 gene expression pattern is calculated as theneighborhood scores (NS) using ACE algorithm.

c) FIG. 11

FIG. 11 depicts an in vivo chemoresistance assay with doxorubicintreatment. Shown are the xenograft tumor sizes from control LM2 orMTDH-KD cells when mice were treated with doxorubicin or drug vehicle.12 tumors per group were used. *P<0.05; **P<0.01 with a two-sidedstudent's t-test to compare KD-1 cells with and without doxorubicintreatment. P=0.022 with Anova analysis of repeated measurement tocompare the whole growth curves of these two conditions.

5. ALDH3A1 and MET Contribute to MTDH-Induced Chemoresistance

Drug uptake and retention assays using paclitaxel and doxorubicin incancer cells with modified MTDH expression revealed that MTDH does notdecrease drug uptake or retention in these cells (FIG. 13). Absent adirect function in altering drug accumulation, MTDH may increasechemoresistance by promoting cellular survival against anti-neoplasticstresses. To further elucidate the molecular mechanism of MTDH-dependentchemoresistance, gene expression profiles of two differentMTDH-knockdown LM2 cell lines were compared with control cells. Asimilar comparison was also performed with LM2 cells co-cultured withHMVEC-L cells (FIG. 12a and Table 7). In the latter analysis, LM2 andHMVEC-L cells were labeled with GFP and the SNARF dye, respectively, toallow FACS-sorting of the two cell populations before RNA extraction(FIG. 12b ). Since MTDH induces significant chemoresistance with orwithout HMVEC-L co-culture, attention was focused on genes that areconsistently present in both conditions (>2.5 fold change in expressionand student's t-test p<0.05). Twenty-three genes (including MTDH) werefound to be under-expressed in MTDH-knockdown cells while 10 genes wereoverexpressed. Among the MTDH down-regulated genes (i.e. genesup-regulated following MTDH knockdown), are the cell death inducinggenes TRAIL and BINP3. TRAIL encodes a TNF family cytokine that inducesapoptosis in tumor cells. Combining TRAIL with conventional anticancerdrugs has been showed to improve therapeutic efficacy ofchemotherapies⁴⁴. BNIP3 is a pro-apoptotic Bcl-2 family gene that hasbeen shown to be involved in apoptotic, necrotic, and autophagic celldeath⁴⁵. Among the MTDH up-regulated genes are several genes previouslyimplicated in chemoresistance of cancer cells, including ALDH3A1, HMOX1,HSP90AB1, HSP90AB3P, and MET. The Hsp90-family heat shock proteins havebeen shown to increase drug resistance by binding and stabilizingP-glycoprotein, which plays a prominent role in multi-drug resistance⁴⁶.The Hsp90 inhibitor geldanamycin increases the sensitivity of resistantcancer cells to cisplatin⁴⁷. Heme oxygenase-1 (HMOX1) is highly inducedby a variety of stress stimuli and cancer chemopreventive agents, andrepresents a prime cellular defense mechanism against oxidative stressvia antioxidant function of its catalytic products. Overexpression ofHMOX1 in human cancers has been shown to confer cellular resistanceagainst chemotherapy and photodynamic therapy⁴⁸. The expression patternof these candidate genes in MTDH knockdown cells was confirmed by qPCRanalysis using samples from both cell cultures and xenograft tumors(FIG. 12c ).

Among these candidate MTDH-downstream genes, ALDH3A1 (aldehydedehydrogenase 3 family, member A1) and MET (hepatocyte growth factorreceptor) are attractive targets due to their physiological functionsand expression patterns. Antineoplastic agents have been shown toproduce oxidative stress in tumors during cancer chemotherapy. Theeffects are mediated, in part, by the generation of aldehydes thatresult from oxidative stress-induced lipid peroxidation. ALDH3A1 encodesan anti-oxidant enzyme with several postulated protective roles thatinclude, but are not limited to, detoxification of peroxidic aldehydesand scavenging of free radicals. Its expression has been implicated inclinical resistance to cyclophosphomide⁴⁹, a mainstay ofchemotherapeutic regimens used to treat breast cancers. Interestingly,as revealed by microarray analysis (FIG. 12a ) and further confirmed byqRT-PCR (data not shown), ALDH3A1 expression is 2 to 3-fold higher inthe HMVEC-L co-culture as compared to the non-co-culture condition,while MTDH knock-down effectively represses ALDH3A1 expression in bothconditions. Such an expression pattern matches the higherchemoresistance of cancer cells induced by HMVEC-L co-culture andchemosensitization by MTDH knock-down in both conditions. To investigatethe functional importance of ALDH3A1 in MTDH-mediated chemoresistance,the LM2 cell line was engineered to express an inducible shRNA againstALDH3A1 to direct the conditional knockdown of ALDH3A1. LM2 cells weremore sensitive to chemotherapeutic agent paclitaxel, doxorubicin and4-hydroxycylcophosphamide (4-HC) when ALDH3A1 knockdown was induced byaddition of doxycycline, while release of ALDH3A1 repression restoredthe chemoresistance of LM2 cells (FIG. 12d ). Furthermore, the abilityof ALDH3A1 to rescue the chemoresistance phenotype in MTDH knockdowncells was examined. Constitutive overexpression of ALDH3A1 in the MTDHknockdown cells was able to partially restore LM2 cell chemoresistanceto paclitaxel and doxorubicin (FIG. 12e ). Together, these resultssuggest that ALDH3A1 is one of the genes that mediate MTDH-inducedchemoresistance.

The chemoresistance function of MET was also examined. In humanpatients, enhanced expression or activation of MET was observed innearly all tumor types. In most cases, its expression is associated bothwith resistance to radiotherapy and chemotherapy, and with poorprognosis⁵⁰. In experimental models, exogenous hepatocyte growth factor(HGF) or overexpression of MET induces resistance to ionizing radiationand many chemotherapeutics, including doxorubicin, cisplatin, etoposide,camptothecin, paclitaxel, TNF and gefitinib in diverse human cancercells from different tumor types, as well as in endothelialcells^(51,52), MET knockdown in LM2 cells lead to a significantreduction of chemoresistance to doxorubicin, an effect that is similarto but weaker than that of MTDH knockdown (FIG. 12f ), indicating thatMET is among MTDH downstream genes that collectively contribute to itsrole in broad-spectrum chemoresistance. Indeed, when MET and ALDH3A1were simultaneously knocked down in LM2 cells, the chemo-sensitizingeffects reached a level comparable to that of MTDH knockdown (FIG. 12f).

a) FIG. 12

FIG. 12 demonstrates that ALDH3A1 and MET contribute to MTDH-mediatedchemoresistance. FIG. 12a depicts expression pattern of the genesregulated in MTDH knockdown cells with or without HMVEC-L co-culture.Some genes previously implicated to promote (red) or suppress (green)cellular chemoresistance were highlighted. FIG. 12b depicts co-culturemicroarray experiment, HMVEC-L were pre-labeled with the SNARF dye andseparated from GFP⁺LM2 cells by FACS before microarray profiling. FIG.12c depicts the confirmation of microarray data by qPCR. Shown areexpression log_(e) (ratio) in LM2 MTDH-KD1 and KD2 cells as compared toLM2 control cells in culture and in xenograft tumors. Genes in red andgreen are those down- or up-regulated in MTDH knockdown cells identifiedby microarray study. FIG. 12d demonstrates that ALDH3A1 knockdownsensitized LM2 cells to chemotherapeutic treatment: (top) ALDH3A1expression levels in cells engineered with ALDH3A1 inducible knockdown,(bottom) clonogenic ability of these cells. FIG. 12e demonstrates thatALDH3A1 overexpression partially rescues the cellular chemoresistance inMTDH knockdown cells: (top) ALDH3A1 expression levels in LM2 cells,(bottom) clonogenic assays. FIG. 12f demonstrates the effect of METknockdown and MET/ALDH3A1 double knockdown on chemoresistance: (top)expression of MET and ALDH3A1 in LM2 cells, (bottom) clonogenic assays.FIG. 12d-f data represent average ±SEM of three replicates. *P<0.05;**P<0.01 with a two-sided student's t-test.

b) FIG. 13

FIG. 13 depicts drug uptake and retention in cells with modified MTDHexpression. Drug update assay of paclitaxel (FIG. 13a ) and doxorubicin(FIG. 13b ) in LM2 parent cells (left panel), LM2 vector control andMTDH knock-down (middle panel), and SCP28 cells with MTDH overexpressionand vector control (right panel). Cells were treated with radiolabeledpaclitaxel or doxorubicin for up to 24 hours and were harvestedimmediately after the indicated period of drug exposure. Drug uptake inthe cells was measured by liquid scintillation counting. Results werenormalized with cellular protein amount measured by Bradford assay andexpressed as average ±SD of three replicates. A drug retention assay forpaclitaxel (FIG. 13c ) and doxorubicin (FIG. 13d ) in various cell linesas in Supplementary FIGS. 13a and 13b was performed. For the retentionstudy, cells were incubated with drug-containing medium for 4 h,followed by incubation in 2 ml drug-free medium for the indicated timeand then harvested. Drug retention in the cells was measured by liquidscintillation counting and normalized with cellular protein amountmeasured by Bradford assay. Results were expressed as percentage ofremaining drugs as compared to the amount at the end of exposure todrug-containing media and shown as average ±SD of three replicates.

6. MTDH Correlates With Poor-Prognosis In Clinical Samples

To evaluate the clinical importance of MTDH in breast cancer, the tissuemicroarrays used in the previous FISH analysis were examined withanti-MTDH antibody. Among the 170 samples on the tissue microarray, 47%expressed MTDH in a moderate to high level (FIG. 14a ). The correlationof MTDH protein levels with 8q22 DNA copy numbers was analyzed using thesamples that exhibited positive immunostaining and FISH results. Whilethe data showed that all but one of the tumors with 8q22 amplificationexpress abundant (medium or high) level of MTDH protein (FIG. 14b ,chi-square test P <0.001), a substantial fraction (12%) of samples withnormal DNA copy numbers also have a high level of MTDH protein.Therefore, alternative mechanisms distinct from 8q22 amplification mayalso result in MTDH activation in breast tumors.

Importantly, MTDH expression is significantly associated with a higherrisk of metastasis (log rank P =0.0058) and shorter survival time(P=0.0008). Univariate survival analysis using the Cox proportionalhazard model also suggested that a high MTDH expression is stronglyassociated with a higher hazard ratio (HR) and worse clinical outcomes(HR=3.7, P=0.01 for metastasis; HR=8.3, P=0.005 for cancer-relateddeath). Immunohistochemical analysis of CCNE2 protein expression(encoded by the only gene present in both poor-prognosis signaturesidentified by van't Veer et al. and Wang et al.) in the same breasttumor tissue array did not reveal any significant correlation withmetastasis (FIG. 15). Interestingly, CCNE2 is located in very closeproximity to the recurrent 8q22 genomic gain (FIG. 15). It is possiblethat the recurrent presence of CCNE2 in multiple poor-prognosissignatures is due to its close physical linkage to 8q22.

To further analyze the prognostic significance of MTDH expressioncompared to other commonly used clinicopathblogical parameters, a Coxhazard ratio analysis of MTDH expression was performed with the tissuesamples stratified by ER, PR, HER2, and p53 status as well as the sizesof primary tumors at the time of cancer diagnosis (Table 8). MTDHexpression level retained its prognostic significance in these analyses,suggesting that it is a prognostic factor independent of otherclinicopathological factors. Indeed, a multivariate Cox analysiscombining all of the above parameters with MTDH expression showed thatthe hazard of metastasis was still significantly higher with MTDHexpressed (P=0.023) even when all the other factors were considered.

a) FIG. 14

FIG. 14 demonstrates that MTDH is associated with poor prognosis ofhuman breast tumors. FIG. 14a depicts MTDH immunostaining with a humanbreast cancer tissue microarray. Shown are typical images of positiveand negative staining. FIG. 14b demonstrates that MTDH protein levelsare positively correlated with the FISH 8q22 DNA copy numbers. FIG. 14cdemonstrates that high MTDH protein level in tumors is associated withearly metastasis in cancer patients. FIG. 14d demonstrates that highMTDH expression is also linked to worse cancer-specific survival. FIG.14e provides a schematic model for the dual role of MTDH in breastcancer progression. In poor-prognosis tumors, 8q22 genomic gain leads tooverexpression of MTDH, which in turn activate two parallel programs topromote chemoresistance and metastasis. Elevated expression ofchemoresistance genes ALDH3A1, MET, HMOX1 and HSP90, as well asrepression of apoptosis inducing genes TRAIL and BNIP3 promote thesurvival and outgrowth of cancer cells in the primary site as well assecondary organs in the face of physiological stress andchemotherapeutic challenges. MTDH additionally promotes metastasis bymediating tumor cell adhesion through the interaction with unknownreceptors and by activating pro-metastasis genes and suppressingmetastasis suppressive genes. Some of the molecular mediators of theMTDH function may play a role in both functional categories. Forexample, MET can promote both metastasis and chemoresistance, andendothelial adhesion can further enhance MTDH-mediated chemoresistance.

b) FIG. 15

FIG. 15 demonstrates that CCNE2 is not associated with clinical outcomesin the breast cancer tissue array analysis. FIG. 15a shows that CCNE2 isthe only overlapping gene in the poor-prognosis gene signatures by van'tVeer et al. and Wang et al., and is located immediately upstream of the8q22 region of gain. Supplementary FIG. 15b shows a human beast cancertissue array was stained with an anti-CCNE2 antibody. A case of highCCNE2 expression (left) and a case of low CCNE2 expression (right) areshown (FIG. 15c ). FIG. 15d depicts Kaplan-Meier analysis of patientmetastasis and survival shows no significance of CCNE2 expression.

-   II. Methods

1. Development of the Analysis of CNAs by Expression (ACE) Algorithm

ACE detects genetic alterations in three steps: 1) calculatingneighborhood scores (NS) for each chromosomal locus as an indicator ofCNA likelihood at that locus, 2) estimating the significance of the NS,and 3) defining the regions of gain and loss. The expression score (ES)for each gene is first calculated according to the correlation of itsexpression with the phenotypes in comparison. Paired t-statistics (forovarian cancer cell lines) or independent t-statistics (for otherdatasets) were used to score gene expression. In general, other metricscan also be used. Consider the genes 1, 2, . . . , N on a chromosomeordered by their physical positions. The NS at locus i was defined asthe weighted sum of the ES of this chromosome:

${NS}_{i} = {\sum\limits_{j = 1}^{N}{w_{ji}{ES}_{j}}}$

where w_(ji) is the weight of gene j. Because the linkage strengthbetween two loci becomes weaker as the distance increases, the weightw_(ji) decreases when locus j is farther way from the locus i. Thecontribution from each gene is weighted by a Gaussian function.

w _(ji)=ce^(−(j−i)) ² ^(/2σ) ²

where c is a constant to normalize all NS into a range of [−1, 1]. Thevariation parameter 2σ² controls the weight decay rate and isarbitrarily set to 100 in the analyses presented here. An analysis usingvarying 2σ² values from 20 to 200 showed similar results with slightshifts at the boundaries of detected regions. For each locus, only thegenes in its physical proximity will have measurable influence on its NSbecause of weight decay. Positive and negative NS suggest genomic gainand loss, respectively. To evaluate the significance of the NS, the genepositions (or sample class labels if the sample size is large enough)are permuted 1,000 times, and each time the NS are recomputed. The pvalues of observed NS are then computed using the distribution ofpermuted NS and adjusted to FDR-q values by the Benjamini-Hochbergprocedure (herein incorporated by reference). In all the CNA analysespresented in this manuscript, a region of genomic gain is defined ashaving at least 20 continuous positive NS of FDR-q <0.01, or a region ofgenomic loss when such NS are all negative. In the epigenetic analysis,a cutoff of 5 continuous NS is used, since epigenetic regulation usuallyhas a smaller functioning range.

Several approaches have been previously reported for CNA predictionbased on expression microarray data^(65,70). The majority of theseapproaches utilized an intuitive “odd-ratio” like method, in which theindividual genes were first defined as significant or not significant bya cutoff of the expression correlation with the phenotype, and thedensities of the significant genes were analyzed for each region with apre-chosen width. The regions with aberrantly high densities werepredicted as regions of gain or loss. Analyses with several expressiondatasets have shown that the “odd-ratio” approach with differentsignificance cutoffs and window sizes generated quite inconsistentresults, and therefore was not suitable for large-scale analysis ofmultiple datasets. ACE can be distinguished from these previousapproaches by several features including: 1) A quantitative expressionscore, instead of the binary significant/non-significant flag of eachgene is used for the regional analysis, which evades the problemassociated with the arbitrary significance cutoff; 2) Aposition-dependent weight is employed for each neighboring gene of thelocus in consideration, which represents a comprehension of the factthat linkage strengths decrease with physical distances; and 3) All thegenes on the chromosome, instead of those within an arbitrarilypre-chosen window size, were analyzed for each genomic locus. Thesefeatures increase the sensitivity and the robustness of the algorithm.

2. Validation of ACE in Various Datasets

To validate the ACE approach, several published expression datasets withthe corresponding information of genomic alterations or long-rangeepigenetic regulation were analyzed. For each expression dataset withprobe detection flags available, the genes that were flagged as “absent”in more than 90% of the samples were removed from further analysis.Duplicate probes mapped to the same transcripts were collapsed and theaverage expression intensities were used. Expression data werenormalized for each study so that each hybridization had equal medianintensity across the entire array. Student's t-test was used to scorethe gene expression prior to NS calculation. To avoid possible bias,dataset-specific optimization of ACE analysis was not performed; auniform set of pre-defined analysis parameters was used instead.

Gene expression data was first analyzed using the Ts1Cje mouse⁷¹, theanimal model for human Down Syndrome and hosts a partial trisomic regionfrom gene Sod1 to Znf295 on chromosome 16. Affymetrix microarrayexpression data of Ts1Cje and normal mouse brain tissues were downloadedfrom the NCBI GEO database (accession number GSE1294). Genes were scoredby the expression difference between trisomic and normal mice followedby NS calculation. ACE detected only one region of gain and no regionsof loss in trisomic mice. The significant region overlapped precisely tothe expected area (FIG. 2a ). The first p-distal boundary in thedetected region corresponds to the gene Mylc2b, which is immediatelyadjacent to Sod1 on the chromosome. The second expected boundary geneZnf295 is located q-distal of all the probes available on the microarrayand ACE consistently defined the region to the end of the q arm.

ACE was then used to analyze gene expression of taxane-resistant ovariancancer cells compared to the parental lines⁷². Results were validatedwith the CGH data for the same samples. The expression and CGH data of 6human ovarian cancer cell lines and their taxane-resistant derivativeswere obtained from the Stanford Microarray Database(http://genome-www5.stanford.edu; herein incorporated by reference). ESwere scored according to the expression difference of each gene betweenthe parental and drug-resistant lines prior to NS calculation. To avoidbias, the same method was used as in the original paper, circular binarysegmentation (CBS)⁷³, to analyze the CGH data. CBS analysis detected 3regions on chromosome 7 with increased copy numbers in thedrug-resistant lines, which was consistent with the previous finding⁷².ACE detected the same areas as the only significant regions (FIG. 2b ).In addition to these significant regions, high concordance was observedbetween the NS and the CBS copy number data throughout the genome. Theoverall correlation between the NS and CGH data was 0.55 (Pearson'scorrelation coefficient), whereas the correlation was only 0.16 if theoriginal expression scores were used, suggesting that NS cansignificantly help uncover the correlation between gene dosage andexpression. From the correlation data, it was determined thatapproximately 30% of all variation observed in NS could be directlyexplained by the underlying variations in genetic copy number³¹.

ACE was further examined using more complicated data from MDA-MB-231cell sublines with different degrees of breast-to-bone metastaticactivities. Expression profiles of 5 highly metastatic lines (2268,2269, 2271, 2274, 2287, 1833) and 5 weakly metastatic lines (2297, 1834,2293, 2295, ATCC) were compared using ACE. This analysis detected 5 CNAevents, including gain at 2p, 6p, 12q, 19q and loss at 7q, inmetastasis. CGH analysis was performed as previously described⁹ on thesecell lines to validate the computational analysis. Four out of these 5genetic events had been directly observed in the cytogenetic analysis.For example, consistent with the ACE prediction, CGH data indicated aloss at the q arm of chromosome 7 in highly metastatic cells (FIG. 2c ).

Long-range epigenetic alteration may also contribute to regional genederegulation. To test ACE's capability to detect such changes, a datasetof 57 bladder tumors⁷⁰ was analyzed; this analysis detected 22 regionswith genes under expressed in tumor tissues as compared to normalsamples. Analysis of the CGH data revealed that 15 of these regions werelost in more than 10% of the tumor tissues, but gained in significantlyfewer tumors (binomial P<0.05), indicating that genomic loss of theseregions was associated with bladder carcinomas. Furthermore, 4 of theremaining regions were proven or suggested by Stransky et al. as regionsunder epigenetic contro1⁷⁰ (Table 4). For example, a region at 3p22.3,was shown to be regulated by histone H3 trimethyl modification in tumorsamples⁷⁰ (FIG. 2d ).

3. Identification of Poor-Prognosis-Associated CNAs in Breast Cancer

Three published breast cancer datasets¹⁴⁻¹⁶ were examined in search ofmetastasis-associated CNAs in breast cancer. The microarray data andpatient records of the tumor samples were obtained from GEO (Wang,GSE2034), and Rosetta websites (van't Veer,http://www.rii.com/publications/2002/vantveer.html); and van de Vijver(http://www.rii.com/publications/2002/nejm.html). Some of the samples inthe van de Vijver study had been previously used in the van't Veerdataset and thus were removed from the van de Vijver dataset to avoidbias. Gene expression was compared between the patients developingmetastasis within 5 years and those free of metastasis for more than 5years. Metastasis-specific CNA regions were identified in each datasetand the SRO regions that were identified in more than one dataset weredefined as the consensus poor-prognosis CNAs. To analyze the prognostic˜10,000 tumors cells was collected in 20 μl of RNAlater stabilizationreagent (Qiagen) for RNA extraction.

DNA extraction was performed as previously described⁷⁴ with or withoutthe paraffin-dissolving step for archived and fresh tumors,respectively. The RNeasy mini kit (Qiagen) was used to extract RNA fromthe tumor samples according to the manufacturer's instructions.

5. Real-Time PCR and Data Evaluation

To analyze the DNA copy numbers, primer pairs were designed using theintron sequences of genes at chromosome 8q, including CA2 (8q21),LAPTM4β and MTDH (8q22), and EIF3S6 (8q23). Real-time PCR and dataanalysis were performed essentially as previously described^(75,76).Briefly, primers were designed using the software PrimerExpress (AppliedBiosystems). PCR was performed using CyberGreen Universal PCR Master Mix(Applied Biosystems) with the ABI Prism 7900HT thermocycler (AppliedBiosystems) according to the manufacturer's protocol. The absolute DNAcopy number of each sample was analyzed with SDS 2.0 software (AppliedBiosystems) using standard curves of known concentrations. The gene APP,located at 21q21 for which no amplifications in breast cancer have beenreported, was used as the internal reference locus^(75,76). The copynumbers of the samples were normalized by healthy human tissue DNA. Thepreviously used copy number ratio threshold 1.8 was applied to define agenomic gain^(75,76).

qRT-PCR was performed to analyze the RNA level of genes at 8q22,including MTDH, LAPTM4β and PTDSS1 in fresh tumors following reversetranscription using the SuperScript first-strand synthesis kit(Invitrogen). The β-actin control kit (Applied Biosystems) was used fornormalization. Primer sequences are listed in Table 9.

6. Fluorescence In Situ Hybridization (FISH)

Tissue FISH was performed by the Dana-Farber Cancer InstituteCytogenetic Core Facility. One microgram of DNA from the BAC cloneRP11-662P7 (Children's Hospital Oakland Research Institute), whichcovers the MTDH locus and other areas at 8q22 was labeled using the NickTranslation kit and SpectrumOrange dUTP (Vysis) following themanufacturer's protocol. Chromosome enumeration probe CEP8 labeled withSpectrumGreen (Vysis) was used for centromere 8 hybridization.Paraffin-embedded tissue slides were pretreated with xylene, dehydratedand digested with Digest-All 3 (Zymed). The slides were then washed in1×PBS, fixed in formalin, and dehydrated in ethanol. Probes were addedonto the slides and denatured at 94° C. for three minutes. Hybridizationwas performed at 37° C. in a humidified chamber. Forty-eight hours laterthe slides were washed in 2×SSC at 72° C. and phosphate bufferedtween-20 solution at room temperature, and counterstained with DAPI.Hybridization signals were viewed on a fluorescence Olympus BX-51microscope system. For each sample 50-100 nuclei were analyzed and theaverage 8q22 copy numbers were calculated. Eighty-two of the 170 sampleson the tissue microarray with successful hybridization were analyzed andscored by the staff of the Cytogenetic Core at Dana Farber CancerInstitute.

7. Generation of Knockdown and Overexpression Cells

MTDH, ALDH3A1 and ME knockdown was achieved with the pSuper-Retro systemwith puromycin or hygromycin selection markers (OligoEngine) using thefollowing sequences: 5′-GGCAGGTATCTTTGTAACTA-3′ (MTDH KD1),5′-GCTGACTGATTCTGGTTCAT-3′ (MTDH KD2), 5′-CGCTACTTATGTGAACGTAA-3′ (MET)and 5′-GGTTCGACCATATCCTGTA-3′ (ALDH3A1). shRNA retroviral vectors weretransfected into the amphotropic Phoenix packaging cell line and viruseswere collected, filtered and used to infect target cells in the presenceof 5 μg/ml polybrene 48 h after transfection. The infected cells wereselected with 0.5 μg/m1 puromycin or/and 0.4 mg/ml hygromycin. Doubleknockdown of MET and ALDH3A1 was achieved by simultaneous infection ofMET and ALDH3A1 targeting viruses with different drug selection markers.MTDH, LAPTM4b, PTDSS1, SDC2, TSPYL5, and UQCRB overexpression wasachieved using the retroviral expression vector pBabe-hygro. Viruseswere generated and used to infect target cells as above and the infectedcells were selected with 0.4 mg/ml hygromycin. For combinationaloverexpression of genes at 8q22, the viruses generated from theexpression vector pBabe-puro containing each of the four genes wereconcentrated by ultracentrifugation and pooled for infection. Northernblots, qRT-PCR, and/or Western blots were performed to validate theknockdown or overexpression of target genes.

To generate an inducible knockdown of ALDH3A1, a retroviral vectorexpressing the Tet repressor (TetR) was constructed by cloning the TetRcoding sequence from the pcDNA6/TR plasmid (Invitrogen) to pQCXIH(Clontech). LM2 cell line with stable expression of TetR was generatedby transduction with retroviruses produced from pQCXIH-TetR. The cellline was then infected with retroviruses generated from the pRSMXvector⁷⁷ containing the ALDH3A1-targeting shRNA sequence. The expressionof a shRNA against ALDH3A1 is under the control of the histone H1promoter and two adjacent tetracycline operators (TetOs). The bacterialTet repressor (TetR) is constitutively expressed from the integratedpQCXIH-TetR in this cell line and suppresses the expression of shRNA bybinding to TetOs. In the presence of 1 μg/ml doxycycline in the media,TetR is released from the TetOs and allow the transcription of ALDH3A1shRNA and thus the repression of ALDH3A1 expression. The pBabe-hygrovector was used to overexpress ALDH3A1.

8. Tumorigenesis and Metastasis Assays in Nude Mice

2×10⁵ cells were washed in PBS and injected intravenously to femaleathymic Ncr-nu/nu mice to study the lung metastasis activity aspreviously described¹¹. For bone metastasis analysis, 1×10⁵ cells wereinjected to the left ventricle of the animal heart as described⁹.Non-invasive bioluminescence imaging was performed to quantify themetastasis burden at the target organs using the IVIS 200 Imaging System(Caliper Life Sciences) as previously described¹¹.

To study primary tumorigenesis, cancer cells harvested from culture wereresuspended in PBS at a concentration of 1×10⁷ cells/ml. An incision wasmade in the abdomen and the skin was recessed to locate the #4 mammaryfat pad, into which 10⁵ cells (10 μl) were injected under a dissectionmicroscope. The primary tumor volume was monitored weekly as previouslydescribed¹¹.

9. In Vivo Chemoresistance Assay

MTDH-knockdown or control LM2 cells (1×10⁶ cells/0.1 ml in a 50:50solution of PBS and Matrigel) were injected subcutaneously into eachflank of nude mice. The mice were treated with chemotherapeutic drugs(20 mg/kg paclitaxel or 5 mg/kg doxorubicin) or the corresponding drugvehicles (Cremophor for paclitaxel and saline for doxorubicin) twice aweek by intravenous delivery a week after the tumor xenografting. Sixmice (12 tumors) were used for each group. Tumor growth was monitoredtwice a week by size measurement. Both maximum (L) and minimum (W)diameters of the tumor were measured using a slide caliper, and thetumor volume was calculated as □LW²/6. Tumor growth was normalized tothat before drug treatment.

10. Lung Histology

Mice were sacrificed and lungs were harvested followed by fixation in10% neural buffered formalin overnight, washing with PBS and dehydrationin 70% ethanol. Tissue paraffin-embedding, sectioning and H&E stainingwere performed by Histoserv, Inc. (Germantown, Md.).

11. Wound Healing Assay

Cancer cells were grown in 10 cm culture dishes to confluence. A“wounding” line was scratched into the cell monolayer using a sterilepipet tip and its width was measured under microscope. The width wasmeasured again at the same place after 3 h of culturing. The migrationdistance was defined as half of the difference between the scratchwidths before and after the culturing period. Six measurements of eachcell line were made and a student's t-test was performed to compare themigration capacity of different cell lines.

12. Two-Chamber Migration Assay

10⁵ luciferase-labeled cancer cells in serum-free medium were seededinto the upper chamber of the insert membranes with a 3 μm pore size (BDBioscience) in a 24-well plate. Serum-containing medium was used in thebottom chamber as the attractant. After 12 h of culturing the cells inthe upper chamber were removed using a cotton swab. The insert membranewith trans-well cells was cut off with a blade and added into a tubewith cell lysis buffer. The cell numbers were quantified using aluciferase assay and the luminescence intensities of each line werenormalized to that of 10⁵ cells. A luciferase signal standard curve ofeach line with 10² to 10⁵ cells was generated for quantification.

13. Matrigel Invasion Assay

Invasion assays were performed essentially as the above migration assayprocedure except that the insert membrane was coated with a Matrigel (BDBioscience) monolayer before cell seeding. Invasion index of each cellline was calculated as the fraction of trans-well cell number divided bythat obtained in the migration assay.

14. Endothelial Adhesion Assay

To test the adhesion of cancer cells to the endothelial cells, differentendothelial cell lines (HBMEC-60, from bone marrow; HUVEC, fromumbilical vein; HMVEC-L, from lung microvascules) and a controlfibroblast cell line WI-38 were grown to confluence in a 24-well plate.10⁵ luciferase-labeled cancer cells were seeded onto the endothelialmonolayer. After 3 h of culturing, the unbound cells in the supernatantwere removed by washing 3 times with PBS and the attached cancer cellswere harvested by trypsinization. The cell number was quantified byluciferase assay as described above.

15. Chemoresistance Clonogenic Assay

Cancer cells with genetic modification of MTDH and/or ALDH3 A1 and thevector control were seeded into a 48-well plate (10⁴ cells/well). After24 h, the cells were treated with apoptosis-inducing chemicals for theindicated time (20, 50 or 100 nM paclitaxel, EMD Biosciences, 24 h; 50,100 or 200 μM doxorubicin, EMD Biosciences, 24 h; 40 μM cisplatin, EMDBiosciences, 2 h; 200 or 500 μM H₂O₂, Fisher Scientific, 2 h) or 10mJ/cm² UV irradiation. After culturing in drug-free DMEM medium foranother 48 h, the surviving cells were quantified by clonogenic assaywith the standard procedure for long-term recovery. Briefly, an aliquotof the harvested cell population was seeded onto a 10 cm dish. Crystalviolet staining was used to count the colonies after 10-day culture inDMEM medium. The colony numbers from untreated cells of the same linewere used to normalize the experimental data. In the HMVEC-L co-cultureassays, HMVEC-L cells were grown to confluence in the 48-well plateswith supplemented EGM-2 medium (Lonza) before seeding of cancer cells.Because HMVEC-L cells could not form colonies in the DMEM medium (datanot shown), the rest of the assay was performed following the standardprocedure.

16. Drug Uptake and Retention Analysis of Paclitaxel and Doxorubicin

Cells were seeded into 12 well plates at densities of 3×10⁵ per well in1 ml of culture medium. One day after seeding, the medium was replacedwith 1 ml of medium containing 50 nM [H³]-Paclitaxel (Moravek, 2Ci/mmol)or 100 nM [C¹⁴]-Doxorubicin (GE HealthCare, 56 mCi/mmol). A pilot studyshowed biphasic kinetics in the uptake and retention of paclitaxel anddoxorubicin in the parent LM2 cells. Based on this data, 4 and 24 h timepoints were selected for comparison of drug uptake and 4 and 12 h forcomparison of retention, in all derivative cell lines. For the uptakestudy, cells were harvested immediately after incubation withdrug-containing medium. For the retention study, cells were incubatedwith drug-containing medium for 4 h, followed by incubation in 2 mldrug-free medium and then harvested. After washing with cold PBS, thepelleted cells were lysed with 200 ul of 0.1N NaOH. An aliquot (5 ul)was used to determine the protein concentration by Bradford assay(Sigma-Aldrich) with BSA as standards. The remaining cell lysates weretransferred to scintillation count vials and mixed with 4 ml ECoScintscintillation fluid (National Diagnostics) and the radioactivity wasmeasured by liquid scintillation counting. A standard curve wasestablished and used to calculate the amount of cell-associated drug.

17. Endothelial Co-Culture, FACS and Microarray Analysis

HMVEC-L cells were grown to confluence in 150 mm culture dishes andwashed once with PBS before SNARF labeling. The cells were cultured inserum-free EGM-2 medium containing 10 μM SNARF (Molecular Probes) at 37°C. for 30 min followed by washing with PBS twice. 2×10⁶ GFP-labeled LM2control or KD1 cells were seeded into the plate in serum-containing DMEMmedium. Cell sorting was performed in the Princeton Flow Cytometry CoreFacility to purify the GFP⁺LM2 cells by using a FACSVantage SE cellsorter (BD Biosceinces) 48 h later (FIG. 12b ). Cells were collected inRNAlater solution (Qiagen) and RNA extracted with RNeasy mini kit(Qiagen). The quality of purified RNA samples was monitored using a 2100bioanalyzer (Agilent) before expression profiling.

To identify genes regulated by MTDH knockdown, RNA samples of LM2control and MTDH-KD cells with or without HMVEC-L co-culture wereanalyzed with the Agilent Whole Human Genome 4×44 k arrays. RNA sampleswere labeled with Cy5 with the Agilent Low RNA Input LinearAmplification Kit and were hybridized with the Cy3-labeled HumanUniversal Reference RNA (Stratagene). Triplicate arrays were performedfor each sample. Arrays were scanned with an Agilent G2565BA scanner andanalyzed with the Agilent Feature Extraction v9.5 software. The Cy5/Cy3ratios were calculated using the feature medium signal and normalized bythe array median. Microarray data were deposited into the NCBI GEOdatabase with an accession number GSE9187. Probes with >2.5 fold changesand student's t-test p values <0.05 in both culturing conditions wereidentified as the MTDH regulated genes. Several significant genes,including ALDH3A1, MET and HMOX1 were randomly selected for qRT-PCRconfirmation with the RNA samples used for microarray analysis. RNAsamples prepared from cells after the same FACS procedure but withoutHMVEC-L co-culture were also analyzed by qRT-PCR to rule out thepossibility that the expression differences were an artifact of thesorting procedure.

18. Tissue Array Immunostaining

A breast cancer tissue microarray composed of 170 primary tumors wasused in the clinical study. At the time of tumor resection, the patientswere at an age of 25 to 49 years (median=40 yrs, SD=4.7yrs). Allpatients in the study were treated with breast conserving surgeryfollowed by radiation therapy to the intact breast. Systemic therapy wasadministered as clinically indicated in accordance with standardclinical practice. Local or regional relapses were defined as clinicallyand histologically documented relapses in the ipsilateral breast orregional nodes. Distant metastases were defined as clinical evidence ofdistant disease based on clinical and/or radiographic findings (Table6).

Immunostaining was performed at the immunohistochemistry core facilityof the Cancer Institute of New Jersey (CINJ) with a rabbit monoclonalanti-MTDH antibody (Invitrogen) and a rabbit polyclonal anti-CCNE2antibody (Imgenex). A BLAST search of the antigen sequence used to raisethe anti-CCNE2 antibody was performed to ensure it does not cross-reactwith other cyclin E family members. Out of the 170 samples, 117 sampleswere stained successfully for MTDH and 133 samples for CCNE2. Eachsample was scored as negative (0), low (1), medium (2), or high (3)according to staining intensities. A Kapan-Meier curve was used tocompare the survival rates of patients with low (scores 0 and 1) andhigh (scores 2 and 3) levels of MTDH or CCNE2. Log rank and Wilcoxontests were used to compare the differences between curves using the SASstatistical software package. To assess whether the MTDH prognosissignificance was associated with the other clinicopathological factors,Cox analysis of MTDH stratified with the expression status of ER, PR,HER or p53 (negative or positive), or the primary tumor sizes (smalleror larger than 2 cm) was performed. Multivariate Cox analysis with allthe parameters in assessment was also undertaken to analyze thedependence of MTDH significance on other parameters.

19. Pharmacologic Data Analysis

The pharmacological dataset was downloaded from the NCl websitehttp://dtp.nci.nih.gov, where the —logGI₅₀ of 42,796 small molecules andnatural products, as well the SNP microarray data were available for 58human tumor cell lines⁴³. GI₅₀ was defined as the drug concentrationnecessary to inhibit cell growth by 50%. The SNP genotyping data wereanalyzed with the CBS algorithm⁷³. A segment mean value of 0.4 was usedas the threshold to define regional gain at the 8q22 region. Fifteen(26%) out of the 58 cell lines were classified as having a gain.Multiple —logGI₅₀ entries of each compound were filtered a5sdescribed⁴³. The compounds were further filtered to exclude those withGI₅₀ data in less than 50 cell lines. This yielded a total of 24,642compounds for further analysis. The logGI₅₀ mean difference of eachcompound in the cells with and without 8q22 gain was calculated, and thesignificance of this difference was estimated by 1,000 permutations ofthe 8q22 status in the cell lines. The numbers of compounds with higherGI₅₀ associated with 8q22 gain were counted by applying a significancethreshold (0.05, 0.01, or 0.001, etc.) of GI₅₀ difference and werecompared to the permutations. Although the Affymetrix U95v2 expressiondata were also available for these cell lines, the only MTDH probeshowed very low signal intensities for all the samples probably due to aprobe failure. Therefore, no further analysis was performed with theMTDH expression data. Instead, the association of 8q22 copy number withgene expression was assessed by calculating a NS from the expression ofgenes in this region for each cell line as described earlier. APearson's correlation coefficient was calculated between the NS and thecopy number.

20. Statistical Analysis

The Kaplan-Meier method was used to estimate survival curves for thepatients and animals. Log rank test and Wilcoxon test were used tocompare the differences between curves. Two-sided Wilcoxon rank test wasperformed to analyze the bioluminescent imaging results in the in vivostudies. A two-sided independent student's t-test without equal varianceassumption was performed to analyze the results of luciferase assays andclonogenic assays.

1-8. (canceled)
 9. A method of treating a chemoresistant cancer in anindividual, comprising administering to the individual an effectiveamount of an agent that selectively prevents the expression of themetadherin gene in the individual.
 10. The method of claim 9, whereinthe agent is an siRNA.
 11. The method of claim 9, wherein the agent isan anti-sense RNA.
 12. The method of claim 9, wherein the agent leads toknock-out of the metaherin gene in the chromosome of the individual. 13.The method of claim 9, wherein the cancer is metastatic.
 14. The methodof claim 13, wherein the cancer is selected from the group consisting ofbreast cancer colon cancer, lung cancer, pancreatic cancer, prostatecancer, bone cancer, blood cancer, brain cancer, and liver cancer. 15.The method of claim 9, further comprising administering to theindividual an effective amount of a chemotherapeutic agent.
 16. Themethod of claim 15, wherein the chemotherapeutic agent is paclitaxel.17. The method of claim 9, wherein the individual is a human individual.18. A method of reducing chemoresistant cancerous cells in an individualtreated with a chemotherapeutic agent, comprising administering to theindividual an effective amount of an agent that selectively prevents theexpression of the metadherin gene in the individual.
 19. The method ofclaim 18, wherein the agent is an siRNA.
 20. The method of claim 18,wherein the agent is an anti-sense RNA.
 21. The method of claim 18,wherein the agent leads to knock-out of the metaherin gene in thechromosome of the individual.
 22. The method of claim 18, wherein theindividual has metastatic cancer.
 23. The method of claim 18, whereinthe individual has a cancer selected from the group consisting of breastcancer colon cancer, lung cancer, pancreatic cancer, prostate cancer,bone cancer, blood cancer, brain cancer, and liver cancer.
 24. Themethod of claim 18, further comprising administering to the individualthe chemotherapeutic agent.
 25. The method of claim 24, wherein thechemotherapeutic agent is paclitaxel.
 26. The method of claim 18,wherein the individual is a human individual.